Immunoglobulin E-specific allergens against leaf in serum of dogs with clinical features of grass leaf allergy.

BACKGROUND: Grass leaf has been suspected of causing immunoglobulin (Ig)E-mediated immediate hypersensitivity reactions in humans and dogs. However, most studies in this area are case-control studies without in vitro data showing the involvement of IgE in the reaction. Laboratory studies have demonstrated the reactivity to a 50-55 kDa protein with clinical signs immediately after contact with grass leaf material. The clinical findings of dogs with atopic-like dermatitis immediately after contact with grass leaf material suggest the involvement of grass leaves as the allergen source. OBJECTIVES: This study was designed to test the IgE-reactivity of grass leaf proteins in dogs with clinical signs and positive scratch test results against grass leaf material. MATERIALS AND METHODS: The serum of 41 patients with a history of allergy and suspected to grass leaf material was immunoblotted against grass leaf extracts from five suspected grass species. The IgE-positive blots were separated with 2D gel electrophoresis and analysed with mass spectrometry (MS). Commercially supplied proteins were used to validate immunoblot activity. RESULTS: The serum of 25 dogs diagnosed with grass dermatitis had positive IgE-specific immunoblot against one or more grass leaf extracts. The MS data indicated a reactive band at 55 kDa to be beta-amylase or RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit (RbLS). All tested dog sera showed IgE-reactivity with beta-amylase and some with RbLS. CONCLUSIONS AND CLINICAL RELEVANCE: Canines with clinical signs of grass-related dermatitis had IgE-reactivity against grass leaf proteins. Serum IgE-reactivity to beta-amylase and RuBisCO large subunit may indicate that these proteins act as allergens, possibly causing pruritus and skin lesions.

Canine dermatitis on contacting grass leaf: A case series.

BACKGROUND: Pruritus is a common clinical sign in dogs for which owners seek veterinary attention. Its diagnosis and treatment are often a source of frustration for the owner and veterinarian. Contact dermatitis is rarely considered, even when lesions are located only on the skin. This report describes the immediate reaction to grass leaf material after skin exposure, with similarities to human protein contact dermatitis. HYPOTHESIS/OBJECTIVE: To describe the history, clinical findings, diagnostic methods, and characteristics of an undescribed canine pruritic dermatitis that is clinically distinct from canine atopic dermatitis and contact dermatitis. ANIMALS: Eighteen pet dogs living in Australia were referred for unresolved pruritic dermatitis. MATERIALS AND METHODS: Retrospective analysis of clinical records from patients with pruritic dermatitis after contacting grass leaves. Findings on skin testing, isolation, challenge, and description of skin lesions are described. RESULTS: Dogs had an inflammatory and pruritic dermatitis affecting the ventral chest, anterior-medial thighs and distal limb, areas that had been in contact with grass, which dogs instinctively avoided. Erythema and erythematous macules and papules were characteristic in the affected skin, inducing coat loss, and thickened pigmented skin. Isolating the dogs from grass resolved their clinical signs and pruritus returned within hours of contact with causative grass. CONCLUSION AND CLINICAL IMPORTANCE: A literature review revealed that this canine dermatitis was similar to human protein contact dermatitis. The impact may be enormous for the treatment of dogs having this disease because the treatment path differs from the therapeutic options of common canine pruritic skin disorders, including the possibility of cure by avoidance.

17ß-estradiol and progesterone effect on human papillomavirus 16 positive cells grown as spheroid co-cultures.

Human papillomavirus (HPV) is the key epidemiologic factor of cervical cancer, but additional cofactors are mandatory. Estrogen has been considered as one of those. Here, the aim was to study the effects of steroid hormones on HPV16 E6-E7, estradiol receptors ERα and ERß, and progesterone receptor (PR) in HPV16-positive cervical carcinoma cell lines SiHa and CaSki grown as epithelial and fibroblast spheroid co-cultures. The spheroid co-cultures were exposured to 17ß-estradiol or progesterone from day 7 onwards. mRNA levels of HPV16 E6-E7, ERα, ERß and PR normalized against GAPDH were analyzed with quantitative reverse transcription-qPCR (RT-qPCR). 17ß-estradiol and progesterone decreased HPV16 E6-E7 mRNA expression in CaSki and increased in SiHA co-cultures. In CaSki co-cultures, ERß expression was blocked after 17ß-estradiol exposure while in SiHa cells it slightly increased ERß expression. PR expression was seen only in CaSki spheroids and it vanished after exposure to steroid hormones. Fibroblasts expressed all three hormone receptors as monolayers but ERß expression decreased and ERα and PR vanished after co-culturing. Cell culturing platform changes both oncogene and hormone receptor expression in HPV16 positive cervical cancer cell lines. This needs to be considered when in vitro results are extrapolated to in vivo situations.

ZAP-70 genotype disrupts the relationship between microbiota and host, leading to spondyloarthritis and ileitis in SKG mice.

OBJECTIVE: The spondyloarthritides share genetic susceptibility, interleukin-23 (IL-23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides. METHODS: BALB/c ZAP-70(W163C) -mutant (SKG) mice, Toll-like receptor 4 (TLR-4)-deficient SKG mice, and wild-type BALB/c mice were housed under specific pathogen-free conditions. SKG and wild-type BALB/c mice were maintained under germ-free conditions, and some of these mice were recolonized with altered Schaedler flora. All of the mice were injected intraperitoneally with microbial ß-1,3-glucan (curdlan). Arthritis, spondylitis, and ileitis were assessed histologically. Microbiome composition was analyzed in serial fecal samples obtained from mice that were co-housed beginning at the time of weaning, using 454 pyrosequencing. Infiltrating cells and cytokines in the peritoneal cavity were measured by flow cytometry and enzyme-linked immunosorbent assay. Cytokine, endoplasmic reticulum (ER) stress marker, and tight junction protein transcription was measured by quantitative real-time polymerase chain reaction. RESULTS: Microbiota content and response to curdlan varied according to whether T cell receptor signal strength was normal or was impaired due to the ZAP-70(W163C) mutation. Curdlan triggered acute inflammation regardless of the presence of the SKG allele or microbiota. However, no or limited microbiota content attenuated the severity of arthritis. In contrast, ileal IL-23 expression, ER stress, lymph node IL-17A production, goblet cell loss, and ileitis development were microbiota-dependent. Ileitis but not arthritis was suppressed by microbiota transfer upon co-housing SKG mice with wild-type BALB/c mice, as well as by TLR-4 deficiency. CONCLUSION: The interaction between immunogenetic background and host microbiota leads to an IL-23-dependent loss of mucosal function, triggering ileitis in response to curdlan.

Smokeless tobacco increases aneuploidy in oral HPV16 E6/E7-transformed keratinocytes in vitro.

BACKGROUND: The scope of this work was to study synergism between human papillomavirus (HPV) infection and tobacco in vitro, both known to be independent risk factors for oral cancer. METHODS: HPV-positive and HPV-negative oral keratinocytes and oral HPV-negative fibroblasts were exposed to smokeless tobacco extract (STE) prepared from the Scandinavian (STE1) and US-type (STE2) snuff. Cell cycle profiles were determined with flow cytometry, and HPV E6/E7 mRNA expression in HPV-positive cells was assayed using RT-qPCR. RESULTS: The exposure of HPV-positive keratinocytes with STE2 increased the number of aneuploid cells from 27% to 80% of which 44% were in S-phase, while none of the diploid cells were in S-phase. The changes after STE1 exposure were less than seen after STE2: from 27% to 31% of which 34% were in S-phase. STE had no effect on HPV16 E6/E7 expression in HPV-positive keratinocytes. In oral spontaneously transformed, HPV-negative keratinocytes, the number of aneuploid cells at G2-M stage increased after STE1 and STE2 exposure from 3% to 9% and 7%, respectively. In HPV-negative oral fibroblasts, the number of cells at G2-M phase increased from 11% to 21% after STE1 and 29% after STE2 exposure. CONCLUSIONS: The effect of STE varied in the cell lines studied. STE2 increased significantly the proportion of aneuploid cells in HPV-positive oral keratinocytes, but not HPV16 E6/E7 expression. This indicates that tobacco products may enhance the effects of HPV 16 and the risk of DNA aneuploidy increasing risk to malignant transformation.

Interleukin-23 mediates the intestinal response to microbial ß-1,3-glucan and the development of spondyloarthritis pathology in SKG mice.

OBJECTIVE: Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70(W163C)-mutant (SKG) mice injected intraperitoneally with ß-1,3-glucan (curdlan). METHODS: Eight weeks after curdlan injection in wild-type or IL-17A(-/-) SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs. RESULTS: In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22. CONCLUSION: In response to systemic ß-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance.

ß-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice.

OBJECTIVE: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 ß-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-ß-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic ß-glucan or mannan exposure.

Increasing mechanical stimulus induces migration of Langerhans cells and impairs the immune response to intracutaneously delivered antigen.

Skin is subjected regularly to mechanical stimulus. Surprisingly, when studying the use of microneedle arrays to introduce antigen into skin, we observed that mechanical stimulus to skin achieved by application of the arrays or a flat metal plate resulted in temporary depletion of Langerhans cells, with the degree of depletion related to the applied stress, whereas no depletion was seen in the interspersed dendritic epidermal T cell population. Further, a significantly impaired immune response to intracutaneous antigen administration was observed in skin recently subjected to mechanical stimulus. This observation may have implications for selection of sites of skin immunisation and for immunogenicity of infections at skin sites routinely subjected to mechanical stimuli.

Nanopatch-targeted skin vaccination against West Nile Virus and Chikungunya virus in mice.

The 'Nanopatch' (NP) comprises arrays of densely packed projections with a defined geometry and distribution designed to physically target vaccines directly to thousands of epidermal and dermal antigen presenting cells (APCs). These miniaturized arrays are two orders of magnitude smaller than standard needles-which deliver most vaccines-and are also much smaller than current microneedle arrays. The NP is dry-coated with antigen, adjuvant, and/or DNA payloads. After the NP was pressed onto mouse skin, a protein payload co-localized with 91.4 + or - 4.1 APC mm(-2) (or 2925 in total) representing 52% of the delivery sites within the NP contact area, agreeing well with a probability-based model used to guide the device design; it then substantially increases as the antigen diffuses in the skin to many more cells. APC co-localizing with protein payloads rapidly disappears from the application area, suggesting APC migration. The NP also delivers DNA payloads leading to cutaneous expression of encoded proteins within 24 h. The efficiency of NP immunization is demonstrated using an inactivated whole chikungunya virus vaccine and a DNA-delivered attenuated West Nile virus vaccine. The NP thus offers a needle-free, versatile, highly effective vaccine delivery system that is potentially inexpensive and simple to use.

The performance of the HPV16 real-time PCR integration assay.

OBJECTIVES: Integration of high risk human papillomavirus (HPV) into the host genome leads to viral oncogene deregulation predisposing to neoplastic progression. Integration can be detected from pap smear or biopsy and use as marker of progressive disease. DESIGN AND METHODS: We have previously developed a highly sensitive real-time PCR method to determine HPV integration frequency and viral load of HPV16 in clinical samples. The test is accurate and sensitive detecting approx. 50 copies of integrated HPV in the sample. RESULTS: We found that a tenfold excess of episomal form to integrated form interferes with the test, regardless the amount of viral DNA. The same was true with background DNA more than 1500 ng in reaction. CONCLUSIONS: Overall, this method is reproducible and suitable for high-throughput screening of clinical samples, but excess episomal copies might mask the integrated form.

Over-expression of topoisomerase IIalpha is related to the grade of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HPV), but does not predict prognosis in cervical cancer or HPV clearance after cone treatment.

OBJECTIVE: One of the pathways leading to cervical cancer is a loss of normal cell cycle control. Topoisomerase IIalpha and IIbeta are important nuclear proteins controlling the G2/M checkpoint, and shown to be over-expressed in many human cancers. Their links to oncogenic human papillomavirus (HPV) types and their prognostic value in cervical cancer are practically unexplored. MATERIAL AND METHODS: As part of our HPV-PathogenISS study, a series of 150 squamous cell carcinomas (SCC) and 152 CIN lesions were examined using immunohistochemical (IHC) staining for topoisomerase IIalpha (topo IIalpha), and tested for HPV using PCR with three primer sets (MY09/11, GP5/GP6, SPF). Follow-up data were available from all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: Topo IIalpha expression increased with increasing grade of CIN (p = 0.0001), with the most dramatic up-regulation upon progression from CIN2 to CIN3 and peaking in SCC (OR 16.23; 95%CI 7.89-33.38). Topo IIalpha up-regulation was also significantly associated with HR-HPV detection in univariate analysis (OR = 3.07; 95%CI 1.70-5.52), but was confounded by the histological grade (Mantel-Haenszel common OR = 1.622; 95%CI 0.782-3.365), and by entering both p16(INK4a) (9) and Survivin (33) in the multivariate regression model. Topo IIalpha did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic factor in cervical cancer in either univariate or multivariate analysis. CONCLUSIONS: Over-expression of topo IIalpha is significantly associated with progression from CIN2 to CIN3, being a late marker of cell proliferation. Its close association with HR-HPV is plausibly explained by the fact that E7 oncoproteins of these HR-HPV (but not LR-HPV) block the normal pRb-mediated inhibition of topo IIalpha by degrading the wild-type Rb.

Two different global gene expression profiles in cancer cell lines established from etiologically different oral carcinomas.

cDNA arrays were used to characterize the gene expression profiles in 6 oral carcinoma cell lines (UT-SCC-10, UT-SCC-14, UT-SCC-37, UT-SCC-54A and UT-SCC-54B, UT-SCC-74) established from 5 patients with different etiological backgrounds, including young patients, classical risk factors and lichen-derived lesions. In addition, 2 human papillomavirus (HPV)-positive cell lines (hypophraryngeal cancer and HPV16 E6/E7-transformed oral keratinocytes) were similarly tested. Two distinct global gene expression profiles with down-regulated and up-regulated patterns were identified, which closely related to the etiologic backgrounds of the primary tumors. Typically in cluster analysis, interferon or interferon-related genes and T- and B-lymphocyte-related genes were up-regulated in lichen-derived carcinoma cell lines. Common to all carcinoma cell lines were 6 genes, which were up- or down-regulated (IgC mu heavy chain constant region, semaphorin, T-cell growth factor, cAMP-dependent protein kinase beta-catalytic subunit, desmocollin 1A/1B precursor and recA-like protein HsRad51). In HPV-positive cell lines, 13 genes were identified with similar down-regulation as shown in our previous studies on HPV-positive genital cell lines. Importantly, all of these genes were also down-regulated in 3 of the 6 oral cancer cell lines. These data suggest that oral carcinomas with different etiological backgrounds can be distinguished by their different global gene expression patterns.

Effect of confluence state and passaging on global cancer gene expression pattern in oral carcinoma cell lines.

BACKGROUND: After establishment of a cell culture, inhomogenous growth and cell selection takes place at early passages. Reproducing the same confluence state among experiments might be difficult. MATERIALS AND METHODS: Here we used cDNA arrays to compare the global gene expression pattern of two oral cancer cell lines at 80%, 100% and super-confluence stages. Also, the stability of the global gene expression pattern during culture was assessed in two cell lines at passages 10 and 53 (early and late passage). An intraclass correlation coefficient test was used for comparisons. RESULTS: The consistency between the different confluence states was almost perfect (> 0.8) and substantial (0.6-0.8). Also the consistency between early and late passages was almost perfect in the cell lines used. CONCLUSION: Our results indicate that neither the state of confluence nor passage have significant effects on the global gene expression profile, despite the variability in expression levels of individual genes.

Divergent expression changes of telomerase and E6/E7 mRNA, following integration of human papillomavirus type 33 in cultured epithelial cells.

Expression of the viral oncogenes E6 and E7, and telomerase, was investigated, using a cell line from a mild dysplastic vaginal lesion containing human papillomavirus (HPV) type 33. During passaging of the cells, there was a change towards a cancer phenotype, and a shift from episomal to integrated HPV. Levels of hTERT (catalytic subunit of telomerase) mRNA, and telomerase activity in cells carrying episomal virus seemed constant during passaging. During passaging of cells containing integrated HPV, however, the levels of oncogene mRNA decreased, while hTERT mRNA and telomerase activity increased sharply. Thus, in those cells there is no direct correlation between changes of oncogene and telomerase expression. Conceivably, viral oncogene expression might trigger telomerase up-regulation in an early subpopulation of cells, which during subsequent passaging could be selected for.

Meconium stimulates neutrophil oxidative burst.

Acute lung injury induced by meconium aspiration is characterized by rapidly developing pulmonary inflammation with influx of activated polymorphonuclear cells. To evaluate the role of meconium in the activation of these invading cells, we described the oxidative capacity of circulating neutrophils after intratracheal administration of thick human meconium in pigs. We also examined the direct effects of varying meconium concentrations on the oxidative burst of human neutrophils in vitro. In neutrophils isolated from meconium-insufflated pigs, phorbol myristate acetate stimulation led to an average 11.7-fold increase in production of reactive oxygen species, measured by chemiluminescence, whereas the increase in control cells from saline-instilled pigs was only 3.1-fold, p =.012 between the groups. Activation of unstimulated human leukocytes by meconium resulted in a dose-dependent response. The lowest meconium concentration (0.2 mg/mL) had an inhibitory effect on neutrophil activation, whereas higher concentrations of meconium (1 and 2 mg/mL) increased neutrophil oxygen radical production progressively. These results thus indicate that moderate and high concentrations of aspirated meconium rapidly activate circulating neutrophils with a resulting oxidative burst contributing to pulmonary tissue injury, whereas low contamination of the aspirated material may in fact suppress the development of oxidative lung injury.

Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration.

Alterations in gene expression represent key events in carcinogenesis. We have studied HPV-induced cervical carcinogenesis, using an HPV-33-positive cell line (UT-DEC-1) established from a low-grade vaginal dysplasia (VAIN-I). Early-passage cells contained HPV-33 in episomal form, but these were superseded at later passages by cells carrying only integrated virus. To gain insight into the biologic significance of HPV integration, we compared the level of gene expression in normal vaginal keratinocytes, early-passage and late-passage UT-DEC-1 cells, using cDNA microarrays. Total RNA was isolated from cells by CsCl-gradient centrifugation, reverse-transcribed with MMLV reverse transcriptase and labeled with alpha-(32)P ATP. A cDNA microarray expression profile analysis was performed with Clontech's Human Cancer 1.2 cDNA expression array kit. The 16 upregulated genes (cut-off 2-fold), identified by comparing both cell types to control keratinocytes, appeared to support cell-cycle progression or to be functional in mitosis. These included, e.g., MCM4 DNA replication licensing factor, cdc2p34 and chromatin assembly factor 1 p48 subunit. Downregulated genes (44 altogether) interfered with apoptosis and cell adhesion, including the apoptosis-inducing genes FRAP, Bik and caspase-9 precursor. The most significant differences between the late and early passages (29 and 46 constantly up- and downregulated genes without any fluctuation) were overexpression of the transcription factors E2F5 with its dimerization partner DP1, NF-kappa B and serine/threonine kinases and underexpression of enzymes of the MAPK pathway. Acquisition of a selective growth advantage after viral integration might be explained by a major shift from a MAPK pathway to cell-cycle dysregulation (G(2)/M).