PGE2 expression by HPV6/11-induced respiratory papillomas blocks NK cell activation in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP), a rare chronic disease caused primarily by human papillomavirus types 6 and 11, consists of repeated growth of premalignant papillomas in the airway. RRP is characterized by multiple abnormalities in innate and adaptive immunity. Natural killer (NK) cells play important roles in immune surveillance and are part of the innate immune responses that help prevent tumor growth. We identified that papillomas lack classical class I MHC and retain nonclassical class I MHC expression. Moreover, in this study, we have identified and characterized the mechanism that blocks NK cell targeting of papilloma cells. Here, we show for the first time that the PGE2 secreted by papilloma cells directly inhibits NK cells activation/degranulation principally through the PGE2 receptor EP2, and to a lesser extent through EP4 signaling. Thus, papilloma cells have a potent mechanism to block NK cell function that likely supports papilloma cell growth.

Role of chemokines in HPV-induced cancers.

Human papillomaviruses (HPVs) cause cancers of the uterine cervix, oropharynx, anus, and vulvovaginal tract. Low-risk HPVs, such as HPV6 and 11, can also cause benign mucosal lesions including genital warts, and in patients with recurrent respiratory papillomatosis, lesions in the larynx, and on occasion, in the lungs. However, both high and less tumorigenic HPVs share a striking commonality in manipulating both innate and adaptive immune responses in HPV- infected keratinocytes, the natural host for HPV infection. In addition, immune/inflammatory cell infiltration into the tumor microenvironment influences cancer growth and prognosis, and this process is tightly regulated by different chemokines. Chemokines are small proteins and exert their biological effects by binding with G protein-coupled chemokine receptors (GPCRs) that are found on the surfaces of select target cells. Chemokines are not only involved in the establishment of a pro-tumorigenic microenvironment and organ-directed metastases but also involved in disease progression through enhancing tumor cell growth and proliferation. Therefore, having a solid grasp on chemokines and immune checkpoint modulators can help in the treatment of these cancers. In this review, we discuss the recent advances on the expression patterns and regulation of the main chemokines found in HPV-induced cancers, and their effects on both immune and non-immune cells in these lesions. Importantly, we also present the current knowledge of therapeutic interventions on the expression of specific chemokine and their receptors that have been shown to influence the development and progression of HPV-induced cancers.

Oropharyngeal tumor cells induce COX-2 expression in peripheral blood monocytes by secretion of IL-1α.

Oropharyngeal squamous cell cancer (OPC) accounts for 3% of all cancers and greater than 1.5% of all cancer deaths in the United States, with marked treatment-associated morbidity in survivors. More than 80% of OPC is caused by HPV16. Tumors induced by HPV have been linked to impaired immune functions, with most studies focused on the local tumor microenvironment. Fewer studies have characterized the effects of these tumors on systemic responses in OPC, especially innate responses that drive subsequent adaptive responses, potentially creating feed-back loops favorable to the tumor. Here we report that elevated plasma levels of PGE2 are expressed in half of patients with OPC secondary to overexpression of COX-2 by peripheral blood monocytes, and this expression is driven by IL-1α secreted by the tumors. Monocytes from patients are much more sensitive to the stimulation than monocytes from controls, suggesting the possibility of enhanced immune-modulating feed-back loops. Furthermore, control monocytes pre-exposed to PGE2 overexpress COX-2 in response to IL-1α, simulating responses made by monocytes from some OPC patients. Disrupting the PGE2/IL-1α feed-back loop can have potential impact on targeted medical therapies.

Toll-like receptor agonists, poly(I:C) and flagellin, lead to IL-36γ induction with divergent release kinetics and differentially alter autophagy in primary human keratinocytes

IL-36γ, a pro-inflammatory member of the IL-1 cytokine superfamily, can be induced and secreted by normal human foreskin keratinocytes (HFKs) in response to pathogenic stimuli, however, the mechanisms underlying the secretion are unknown. In this study, we demonstrate that stimulation with the TLR3 agonist, poly (I:C), led to a delayed secretion of IL-36γ compared to stimulation with the TLR5 agonist, flagellin, despite equal levels of the cytokine (p = 0.006). IL-36γ was shown to be released from HFKs in its inactive, uncleaved form, based on western blotting. Moreover, recombinant IL-36γ in its activated, cleaved form induced endogenous IL-36γ 10-fold (p = 0.004) and CXCL8 five-fold (p = 0.003) over baseline levels compared to unactivated full-length recombinant IL-36γ. The ratio of LC3b-II/LC3b-I was significantly higher in poly(I:C)-treated cells compared to flagellin-treated and unstimulated controls without a change in SQSTM1/p62 after 24 hours of stimulation (p = 0.043). Under fluorescence microscopy, poly(I:C) led to a two-fold increase at eight hours and four-fold increase at 24 hours in accumulated autophagosomes post-stimulation (p = 0.032). In contrast, autophagosomes were unchanged relative to baseline in response to flagellin. Bafilomycin A1 treatment enhanced poly(I:C)-mediated IL-36γ secretion (p = 0.044) while rapamycin led to a noticeable, but non-significant, increase in flagellin-mediated IL-36γ secretion, indicating that interrupting autophagic flux can alter IL-3γ grelease from HFKs. Finally, we show that, compared to clinically normal laryngeal tissue, there were significantly higher levels of LC3b-II in HPV-infected respiratory papilloma tissue, indicating a higher number of autophagosomes; a signature of disrupted autophagic flux.

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells.

Determinants of the lung microbiome in intubated premature infants at risk for bronchopulmonary dysplasia.

BACKGROUND: Airway dysbiosis in premature infants may be associated with bronchopulmonary dysplasia (BPD). Early oropharyngeal colostrum (OPC) administration alters the oral microbiome, which may impact the lung microbiome. We aim to compare the oral and tracheal microbiota during the first week of life, and to determine whether early OPC administration affects microbial diversity or leukocyte inflammatory activity in the lung. METHODS: Intubated premature infants (n = 42) were evaluated. The oral microbiome was characterized on day of life (DOL) 3, and the tracheal microbiome on DOL 3 and DOL 7, using 16S ribosomal DNA sequencing. Gene expression for inflammatory markers was quantified in airway leukocytes by real-time q-PCR. RESULTS: The oral and tracheal microbiota were significantly different on DOL 3, but the tracheal microbiome on DOL 7 was more similar to the oral from DOL 3. Tracheal bacterial diversity decreased from DOL 3 to DOL 7. Longer time to first OPC administration tended to be associated with lower bacterial diversity in the airways. CONCLUSIONS: The tracheal microbiome in intubated premature infants in the first week is likely determined, in part, by the composition of the oral microbiome. Bacterial diversity in intubated babies decreases during the first week of life, a pattern that could be consistent with risk for BPD. Decreased bacterial diversity and increased inflammatory activity in the lung may also be associated with delayed administration of OPC.

A novel cancer preventative botanical mixture, TriCurin, inhibits viral transcripts and the growth of W12 cervical cells harbouring extrachromosomal or integrated HPV16 DNA.

BACKGROUND: The phytochemical mixture TriCurin (curcumin, epigallocatechin gallate (EGCG) and resveratrol) eliminates human papillomavirus (HPV) (+) cancer cells in vitro and in vivo. In this study, we further evaluate TriCurin. METHODS: The activity of TriCurin and its individual compounds was assayed on W12 cells, derived from a cervical precancer containing episomal and integrated HPV16 DNA, using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, microscopy and reverse transcription-polymerase chain reaction (RT-PCR), and on HeLa cells by gene expression analysis. The stability and toxicity of TriCurin microemulsion were tested in an organotypic cervical tissue model. RESULTS: TriCurin and its individual compounds inhibit the growth of W12 cells, episomal, type 1 and 2 integrants; the relative order of activity is TriCurin, EGCG, curcumin, or resveratrol. RT-PCR shows that TriCurin activates p53 and suppresses HPV16 mRNAs E1, E2, E4, E6 and E7 at 24 h in W12 cells. Gene expression analysis shows that TriCurin activates pro-apoptotic genes and represses anti-apoptotic genes in HeLa cells. TriCurin in a microemulsion is stable and non-toxic to cervical tissue. The combination of TriCurin and tanshinone IIA exhibits additional synergy against HeLa cells. CONCLUSIONS: TriCurin, and the combination of TriCurin with tanshinone IIA, are effective against HPV (+) cells. The phytochemical mixture, in the microemulsion-based cream, is a promising therapeutic for the prevention and treatment of cervical cancer.

Altered Monocyte and Langerhans Cell Innate Immunity in Patients With Recurrent Respiratory Papillomatosis (RRP).

The micromilieu within respiratory papillomas supports persistent human papillomavirus (HPV) infection and disease recurrence in patients with recurrent respiratory papillomatosis (RRP). These patients show polarized (TH2-/Treg) adaptive immunity in papillomas and blood, enriched immature Langerhans cell (iLC) numbers, and overexpression of cyclooxygenase-2/prostaglandin E2 (PGE2) in the upper airway. Blood monocyte-derived, and tissue-derived iLCs from RRP patients and controls were now studied to more fully understand innate immune dysregulation in RRP. Patients' monocytes generated fewer iLCs than controls, due to a reduced fraction of classical monocytes that generated most but not all the iLCs. Prostaglandin E2, which was elevated in RRP plasma, reduced monocyte-iLC differentiation from controls to the levels of RRP patients, but had no effect on subsequent iLC maturation. Cytokine/chemokine responses by iLCs from papillomas, foreskin, and abdominal skin differed significantly. Freshly derived tissue iLCs expressed low CCL-1 and high CCL-20 mRNAs and were unresponsive to IL-36γ stimulation. Papilloma iLCs uniquely expressed IL-36γ at baseline and expressed CCL1 when cultured overnight outside their immunosuppressive microenvironment without additional stimulation. We conclude that monocyte/iLC innate immunity is impaired in RRP, in part due to increased PGE2 exposure in vivo. The immunosuppressive papilloma microenvironment likely alters iLC responses, and vice versa, supporting TH2-like/Treg HPV-specific adaptive immunity in RRP.

Microarray analysis of human keratinocytes from different anatomic sites reveals site-specific immune signaling and responses to human papillomavirus type 16 transfection.

BACKGROUND: Stratified human keratinocytes (SHKs) are an essential part of mucosal innate immune response that modulates adaptive immunity to microbes encountered in the environment. The importance of these SHKs in mucosal integrity and development has been well characterized, however their regulatory immunologic role at different mucosal sites, has not. In this study we compared the immune gene expression of SHKs from five different anatomical sites before and after HPV16 transfection using microarray analyses. METHODS: Individual pools of human keratinocytes from foreskin, cervix, vagina, gingiva, and tonsils (HFKs, HCKs, HVKs, HGKs and HTLKs) were prepared. Organotypic (raft) cultures were established for both normal and HPV16 immortalized HFKs, HCKs, HVKs, HGKs and HTLKs lines which stably maintained episomal HPV16 DNA. Microarray analysis was carried out using the HumanHT-12 V4 gene chip (Illumina). Immune gene expression profiles were obtained by global gene chip (GeneSifter) and Ingenuity pathway analysis (IPA) for each individual site, with or without HPV16 transfection. RESULTS: We examined site specific innate immune response gene expression in SHKs from all five different anatomical sites before and after HPV16 transfection. We observed marked differences in SHK immune gene repertoires within and between mucosal tracts before HPV 16 infection. In addition, we observed additional changes in SHKs immune gene repertoire patterns when these SHKs were productively transfected with HPV16. Some immune response genes were similarly expressed by SHKs from different sites. However, there was also variable expression of non-immune response genes, such as keratin genes, by the different SHKs. CONCLUSIONS: Our results suggest that keratinocytes from different anatomical sites are likely hard wired in their innate immune responses, and that these immune responses are unique depending on the anatomical site from which the SHKs were derived. These observations may help explain why select HPV types predominate at different mucosal sites, cause persistent infection at these sites, and on occasion, lead to HPV induced malignant and benign tumor development.

Poly(I:C) induces controlled release of IL-36γ from keratinocytes in the absence of cell death.

The epithelium is part of an integrated immune system where cytokines, toll-like receptors and their ligands, and extracellular vesicles play a crucial role in initiating an innate immune response. IL-36γ is a pro-inflammatory member of the IL-1 family that is mainly expressed by epithelial cells, but regulation of its expression and release are only beginning to be understood. Previous studies reported that IL-36γ is abundant in recurrent respiratory papillomatosis, a rare but devastating disease caused by human papillomaviruses (HPV) types 6 and 11, in which papillomas recurrently grow in and block the airway. Despite the overexpression of IL-36γ, papilloma tissues show no evidence of inflammation, possibly due to suppression of its release by HPVs. We have used primary human foreskin keratinocytes as a model to study IL-36γ regulation in normal epithelial cells. Low doses of poly(I:C) mediate expression and release of IL-36γ without inducing the cell death reported by those using high doses. PKR, an enzyme required for inflammasome activation, does not contribute to controlled release of IL36γ. The keratinocytes secrete IL-36γ in two forms, soluble and in extracellular vesicles. We conclude that there are two separately regulated pathways for the controlled secretion of IL-36γ from keratinocytes, which could contribute to the modulation of both local and systemic immune responses to viruses and other pathogens.

Immune Dysregulation in Patients Persistently Infected with Human Papillomaviruses 6 and 11.

Human Papillomaviruses (HPVs) 6 and 11 are part of a large family of small DNA viruses, some of which are commensal. Although much of the population can contain or clear infection with these viruses, there is a subset of individuals who develop persistent infection that can cause significant morbidity and on occasion mortality. Depending on the site of infection, patients chronically infected with these viruses develop either recurrent, and on occasion, severe genital warts or recurrent respiratory papillomas that can obstruct the upper airway. The HPV-induced diseases described are likely the result of a complex and localized immune suppressive milieu that is characteristic of patients with persistent HPV infection. We review data that documents impaired Langerhans cell responses and maturation, describes the polarized adaptive T-cell immune responses made to these viruses, and the expression of class select II MHC and KIR genes that associate with severe HPV6 and 11 induced disease. Finally, we review evidence that documents the polarization of functional TH2 and T-regulatory T-cells in tissues persistently infected with HPV6 and 11, and we review evidence that there is suppression of natural killer cell function. Together, these altered innate and adaptive immune responses contribute to the cellular and humoral microenvironment that supports HPV 6 and 11-induced disease.

Decreased Langerhans cell responses to IL-36γ: altered innate immunity in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare, chronic disease caused by human papillomaviruses (HPVs) types 6 and 11 that is characterized by the polarization of adaptive immune responses that support persistent HPV infection. Respiratory papillomas express elevated mRNA levels of IL-36γ, a proinflammatory cytokine in comparison to autologous clinically normal laryngeal tissues; however there is no evidence of inflammation in these lesions. Consistent with this, respiratory papillomas do not contain TH1-like CD4(+) T-cells or cytotoxic CD8(+) T-cells, but instead contain a predominance of TH2-like and T regulatory cells (Tregs). In addition, papillomas also are infiltrated with immature Langerhans cells (iLCs). In this study, we show that papilloma cells express IL-36γ protein, and that human keratinocytes transduced with HPV11 have reduced IL-36γ secretion. We now provide the first evidence that peripheral blood-derived iLCs respond to IL-36γ by expressing inflammatory cytokines and chemokines. When stimulated with IL-36γ, iLCs from patients with RRP had lower expression levels of the TH2-like chemokine CCL-20 as compared with controls. Patients' iLCs also had decreased steady state levels of CCL-1, which is a proinflammatory chemokine. Moreover, CCL-1 levels in iLCs inversely correlated with the severity of RRP. The combined decrease of TH1- and a TH2-like chemokines by iLCs from patients could have consequences in the priming of IFN-γ expression by CD8(+) T-cells. Taken together, our results suggest that, in RRP, there is a defect in the proinflammatory innate immune responses made by iLCs in response to IL-36γ. The consequence of this defect may lead to persistent HPV infection by failing to support an effective HPV-specific, TH1-like and/or Tc1-like adaptive response, thus resulting in the predominant TH2-like and/or Treg micromilieu present in papillomas.

T(H)2-like chemokine patterns correlate with disease severity in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP), characterized by the recurrent growth of benign tumors of the respiratory tract, is caused by infection with human papillomavirus (HPV), predominantly types 6 and 11. Surgical removal of these lesions can be required as frequently as every 3 to 4 wks to maintain a patent airway. There is no approved medical treatment for this disease. In this study, we have characterized the T(H)2-like chemokine profile (CCL17, CCL18, CCL20, CCL22) in patients with RRP and asked whether it was modulated in patients who had achieved significant clinical improvement. CCL17, CCL18 and CCL22 messenger RNAs (mRNAs) were increased in papillomas compared with clinically normal laryngeal epithelium of the RRP patients. Overall, CCL20 mRNA expression was not increased, but there was intense, selective CCL20 protein expression in the basal layer of the papillomas. Patients with RRP expressed more CCL17 (p = 0.003), CCL18 (p = 0.0003), and CCL22 (p = 0.007) in their plasma than controls. Plasma CCL18 decreased over time in three patients enrolled in a pilot clinical trial of celecoxib, and the decrease occurred in conjunction with clinical improvement. There was a significant correlation between sustained clinical remission in additional patients with RRP and reduced levels of CCL17 (p = 0.01), CCL22 (p = 0.002) and CCL18 (p = 0.05). Thus, the change in expression of these three plasma T(H)2-like chemokines may, with future studies, prove to serve as a useful biomarker for predicting disease prognosis.

Immune suppression in premalignant respiratory papillomas: enriched functional CD4+Foxp3+ regulatory T cells and PD-1/PD-L1/L2 expression.

PURPOSE: Respiratory papillomas, caused by human papillomaviruses types 6 and 11 (HPV6/11), are premalignant lesions with potential for malignant conversion. The cytokine and chemokine micromilieu of papillomas is T(H)2-like with a marked absence of IFN-γ expression. To illuminate why patients with recurrent respiratory papillomatosis (RRP) fail to effectively control their disease, we further investigated the suppressive cellular microenvironment in papillomas. EXPERIMENTAL DESIGN: CD4(+)CD25(+)CD127(low/-)Foxp3(+) regulatory T cells (Treg) and CD4(+)CD25(-)CD127(low/-)Foxp3(-) T cells within papillomas were characterized and isolated. Their suppressor function was measured by inhibition of peripheral blood mononuclear cell (PBMC) proliferation. Expression of PD-1, CD69, and Helios was identified on these T cells. PD-L1, PD-L2, CCL17, and CCL22 mRNA was also identified in papillomas by quantitative PCR. RESULTS: Functional Tregs were markedly enriched in papillomas and strongly inhibited anti-CD3 and anti-CD28 antibody activated PBMC proliferation. The natural Treg marker Helios was reduced on Tregs from papillomas, indicating that the majority of Tregs in papillomas are adaptive. The majority of the papilloma-derived CD4(+) T cells expressed the CD4(+)CD25(-)CD127(low/-)Foxp3(-)PD1(+)CD69(+) phenotype and failed to suppress PBMC proliferation, suggesting that they are chronically activated and exhausted. The Treg-attracting chemokine CCL22 was equally expressed by all laryngeal tissues examined. However, CCL17 was robustly expressed by papillomas compared with unaffected laryngeal tissues from RRP patients and individuals without RRP. PD-L1 was elevated in papillomas compared with control laryngeal tissues. CONCLUSIONS: Papilloma CD4(+) T cells are enriched with functional Tregs, and the adaptive Helios(-) Treg fraction was increased within the T(H)2-like papilloma micromilieu. CD4(+)CD25(-)CD127(low/-)Foxp3(-) T-cells failed to suppress PBMC proliferation and may be exhausted. The PD-1/PDL-1 pathway may represent an additional immunosuppressive mechanism that contributes to defective HPV6/11 clearance in RRP.

Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.

Activating killer cell immunoglobulin-like receptors 3DS1 and 2DS1 protect against developing the severe form of recurrent respiratory papillomatosis.

The polymorphic killer cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell response against viral infection and tumor transformation. Here we investigated if select KIR genes are associated with recurrent respiratory papillomatosis (RRP), a rare disease of the larynx and upper airway caused by human papillomaviruses (HPV)-6/11. DNA from 66 RRP patients and 195 healthy controls were characterized for KIR and HLA gene polymorphism. Patients lacking activating KIR genes 3DS1 and 2DS1 were more common in severe RRP compared with mild-moderate RRP (78.8% vs 48.5%, p = 0.019). Further, patients carrying any of the known susceptible HLA-DRB1/DQB1 alleles were more frequently negative for KIR3DS1 (p = 0.006), KIR2DS1 (p = 0.003) or KIR2DS5 (p = 0.004) compared with controls carrying any of these HLA allotypes. Nearly 80% of the patients with severe disease were missing the protective HLA-DQB1*0602 allele as well as both KIR3DS1 and KIR2DS1 genes. Phenotyping of papilloma-infiltrating mononuclear-cells revealed an elevated numbers of NK cells and CD57(+)CD4(+) T cells in KIR3DS1(-)KIR2DS1(-) patients compared with patients carrying either one or both of these KIRs. Our data suggest that NK cells expressing activating receptors KIR3DS1 and KIR2DS1 may be necessary to trigger an effective early immune response against HPV-infected targets to establish resistance to RRP development.

Cyclooxygenase 2 mediates the antiangiogenic effect of rapamycin in Ewing sarcoma.

BACKGROUND: Rapamycin can inhibit tumor growth and angiogenesis in various human cancers. Cyclooxygenase 2 (COX-2) is involved in the angiogenic process. We hypothesized that the antiangiogenic effect of rapamycin may be mediated by suppression of COX-2. METHODS: Ewing sarcoma (ES) cells were implanted in athymic mice. Selected animals were treated with rapamycin for 5 weeks. Tumor vascularity was assessed by lectin perfusion angiography and immunohistochemistry. Phosphorylation of mammalian target of rapamycin pathway proteins was determined by Western blot analysis. Staining of COX-2 protein was determined by immunohistochemistry, and expression of COX-2 messenger RNA levels was assessed with quantitative real-time (RT) polymerase chain reaction. RESULTS: Mean tumor weights were significantly reduced in the treated group (5.43 g +/- 1.43 SEM vs 0.49 g +/- 0.15 SEM, P < .003). There was abundant vasculature in the control group and blunted vascularity in the treated xenografts. The phosphorylation of p70s6k and Akt was not inhibited in the rapamycin-treated tumors. Cyclooxygenase 2 was suppressed in the treated xenografts at both the protein and messenger RNA levels. CONCLUSION: Low-dose rapamycin inhibits tumor growth and angiogenesis in human ES without inhibiting the phosphorylation of p70s6k and Akt. Cyclooxygenase 2 levels are inhibited by low-dose treatment of ES with rapamycin. Cyclooxygenase 2 suppression may mediate the antiangiogenic effect of rapamycin in Ewing sarcoma.

Alternate splicing of interleukin-1 receptor type II (IL1R2) in vitro correlates with clinical glucocorticoid responsiveness in patients with AIED.

Autoimmune Inner Ear Disease (AIED) is poorly characterized clinically, with no definitive laboratory test. All patients suspected of having AIED are given glucocorticoids during periods of acute hearing loss, however, only half initially respond, and still fewer respond over time.We hypothesized that AIED is a systemic autoimmune disease characterized by dysfunctional peripheral blood mononuclear cells (PBMC) responses to a unique cochlear antigen(s). To test this hypothesis, we examined end-stage AIED patients undergoing cochlear implant surgery and compared autologous perilymph stimulated PBMC from AIED patients to controls. We determined that autologous perilymph from AIED patients was unable to induce expression of a long membrane-bound Interleukin-1 Receptor Type II (mIL1R2) transcript in PBMC as compared with controls, despite similar expression of the short soluble IL1R2 (sIL1R2) transcript (p<0.05). IL1R2 is a molecular decoy that traps interleukin-1beta (IL-1beta) and does not initiate subsequent signaling events, thereby suppressing an inflammatory response. IL1R2 transcript length is regulated by alternate splicing, and the major inhibitory function is attributed to the full-length mIL1R2. In addition, IL1R2 expression is induced by dexamethasone.Separately, we prospectively examined patients with newer onset glucocorticoid-responsive AIED. Immediately prior to clinical treatment for acute deterioration of hearing thresholds, their PBMC demonstrated a robust induction of mIL1R2 in PBMC in response to dexamethasone in vitro that correlated with a clinical response to prednisone in vivo (p<0.0001) as measured by hearing restoration. In contrast, clinically steroid unresponsive patients demonstrated high basal levels of mIL1R2 in their PBMC and only minimally augmented expression in response to dexamethasone. Thus, induced expression of mIL1R2 appears to be a protective mechanism in hearing homeostasis and warrants further investigation in a large prospective clinical trial to determine if IL1R2 can be used as a specific biomarker for AIED.

Immune dysregulation and tumor-associated gene changes in recurrent respiratory papillomatosis: a paired microarray analysis.

Recurrent respiratory papillomas (RRP) are benign airway tumors, caused primarily by human papillomaviruses (HPV) types 6 and 11. The disease is characterized by multiple recurrences after surgical removal, with limited effective therapy. To identify novel targets for future therapy, we established transcriptional profiles for actively growing papillomas compared with autologous, clinically normal, laryngeal epithelia (adjacent tissue). Total ribonucleic acid (RNA) from 12 papillomas and 12 adjacent tissues were analyzed by microarray, and the matched sets of tissues compared by paired t test, to identify differentially expressed genes in papilloma tissues while minimizing variations intrinsic to individual patients. Quantitative polymerase chain reaction (PCR) was used to confirm the relative expression levels for a subset of genes. Within the 109 differentially expressed transcripts whose expression varied at least three-fold were two large groups of genes with related functions. The first group consisted of 18 genes related to host defense, including both innate and adaptive immunity. The second group contained 37 genes that likely contribute to growth of papillomas as benign tumors, since the altered pattern of expression also had been reported previously in many cancers. Our results support our previous studies that document a systemic T(H)2-like adaptive immune response in RRP, and suggest that there is a role for altered innate immunity in RRP as well. We propose that HPV 6 and 11 infection establishes a tumorigenic microenvironment characterized by alteration of both innate inflammatory signals and adaptive immune responses that prevent effective T(H)1-like response, in conjunction with altered expression of numerous genes that regulate cellular growth and differentiation.