Relationship between gene expression patterns from nasopharyngeal swabs and serum biomarkers in patients hospitalized with COVID-19, following treatment with the neutralizing monoclonal antibody bamlanivimab.

BACKGROUND: A thorough understanding of a patient's inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. METHODS: 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. RESULTS: Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. CONCLUSIONS: Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.

Erythromycin reduces nasal inflammation by inhibiting immunoglobulin production, attenuating mucus secretion, and modulating cytokine expression.

Allergic rhinitis (AR) and chronic rhinosinusitis (CRS) share some similar pathological mechanisms. In current study, we intend to investigate the impact of AR on CRS. In addition, we explored the efficacy of erythromycin (EM) treatment on CRS mice with or without AR (CRSwoAR, CRSwAR). Study subjects were divided into control, CRSwoAR, and CRSwAR groups. Experimental mice were divided similarly into control, CRSwoAR, and CRSwAR groups. In addition, CRS mice were treated with EM at 0.75, 7.5, or 75 mg/kg or with dexamethasone (Dex) at 1 mg/kg. In our results, allergy exacerbates inflammation that was evident in nasal histology and cytokine expression both in patients and in mice with CRS. Dex 1 mg/kg, EM 7.5 or 75 mg/kg treatments significantly inhibited serum IgE and IgG2a in CRS mice. EM-treated CRS mice had significantly elevated IL-10 levels and had a reversal of Th-1/Th-2 cytokine expression in nasal-associated lymphoid tissue. MUC5AC expressions were significantly reduced in the 7.5 or 75 mg/kg EM-treated mice compared with untreated mice. EM showed inhibitions on immunoglobulin production and mucus secretion stronger than Dex. We concluded that comorbid AR enhanced inflammation of CRS. EM and Dex treatments showed similar anti-inflammatory effects on CRS but through partly different mechanisms.

Orchestration of myeloid-derived suppressor cells in the tumor microenvironment by ubiquitous cellular protein TCTP released by tumor cells.

One of most challenging issues in tumor immunology is a better understanding of the dynamics in the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TIME), as this would lead to the development of new cancer therapeutics. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME via activation of Toll-like receptor-2. As further proof of principle, we show that inhibition of TCTP suppresses PMN-MDSC accumulation and tumor growth. In human cancers, we find an elevation of TCTP and an inverse correlation of TCTP gene dosage with antitumor immune signatures and clinical prognosis. This study reveals the hitherto poorly understood mechanism of the MDSC dynamics in the TIME, offering a new rationale for cancer immunotherapy.

Insights into adult atopic dermatitis heterogeneity derived from circulating biomarker profiling in patients with moderate-to-severe disease.

Atopic dermatitis (AD) is a heterogeneous systemic inflammatory skin disease associated with dysregulated immune responses, barrier dysfunction and activated sensory nerves. To characterize circulating inflammatory profiles and underlying systemic disease heterogeneity within AD patients, blood samples from adult patients (N = 123) with moderate-to-severe AD in a phase 2 study of baricitinib (JAHG) were analysed. Baseline levels of 131 markers were evaluated using high-throughput and ultrasensitive proteomic platforms, patient clusters were generated based on these peripheral markers. We implemented a novel cluster reproducibility method to validate cluster outcomes within our study and used publicly available AD biomarker data set (73 markers, N = 58 patients) to validate our findings. Cluster reproducibility analysis demonstrated best consistency for 2 clusters by k-means, reproducibility of this clustering outcome was validated in an independent patient cohort. These unique JAHG patient subgroups either possessed elevated pro-inflammatory mediators, notably TNFß, MCP-3 and IL-13, among a variety of immune responses (high inflammatory) or lower levels of inflammatory biomarkers (low inflammatory). The high inflammatory subgroup was associated with greater baseline disease severity, demonstrated by greater EASI, SCORAD Index, Itch NRS and DLQI scores, compared with low inflammatory subgroup. African-American patients were predominantly associated with the high inflammatory subgroup and increased baseline disease severity. In patients with moderate-to-severe AD, heterogeneity was identified by the detection of 2 disease subgroups, differential clustering amongst ethnic groups and elevated pro-inflammatory mediators extending beyond traditional polarized immune responses. Therapeutic strategies targeting multiple pro-inflammatory cytokines may be needed to address this heterogeneity.

Predictive biomarker modeling of pediatric atopic dermatitis severity based on longitudinal serum collection.

The pathogenesis of atopic dermatitis (AD) results from complex interactions between environmental factors, barrier defects, and immune dysregulation resulting in systemic inflammation. Therefore, we sought to characterize circulating inflammatory profiles in pediatric AD patients and identify potential signaling nodes which drive disease heterogeneity and progression. We analyzed a sample set of 87 infants that were at high risk for atopic disease based on atopic dermatitis diagnoses. Clinical parameters, serum, and peripheral blood mononuclear cells (PBMCs) were collected upon entry, and at one and four years later. Within patient serum, 126 unique analytes were measured using a combination of multiplex platforms and ultrasensitive immunoassays. We assessed the correlation of inflammatory analytes with AD severity (SCORAD). Key biomarkers, such as IL-13 (rmcorr=0.47) and TARC/CCL17 (rmcorr=0.37), among other inflammatory signals, significantly correlated with SCORAD across all timepoints in the study. Flow cytometry and pathway analysis of these analytes implies that CD4 T cell involvement in type 2 immune responses were enhanced at the earliest time point (year 1) relative to the end of study collection (year 5). Importantly, forward selection modeling identified 18 analytes in infant serum at study entry which could be used to predict change in SCORAD four years later. We have identified a pediatric AD biomarker signature linked to disease severity which will have predictive value in determining AD persistence in youth and provide utility in defining core systemic inflammatory signals linked to pathogenesis of atopic disease.

Characterization of the cytokine storm reflects hyperinflammatory endothelial dysfunction in COVID-19.

BACKGROUND: Physicians treating patients with coronavirus disease 2019 (COVID-19) increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared with healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. OBJECTIVE: To identify and characterize the host inflammatory response to severe acute respiratory syndrome coronavirus 2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease in patients stratified as mild, moderate, and severe versus matched healthy controls. METHODS: Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. RESULTS: Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within patients with COVID-19, and strong biomarker association with patient response as measured by Ordinal Scale. As patients progress, we observe statistically significant dysregulation of IFN-γ, IL-1RA, IL-6, IL-10, IL-19, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, CXCL9, CXCL10, CXCL5, ENRAGE, and poly (ADP-ribose) polymerase 1. Furthermore, in a limited series of patients who were sampled frequently, confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. CONCLUSIONS: These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of severe acute respiratory syndrome coronavirus 2 pathogenesis and the host response.

A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian Cancer.

Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by selected reaction monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes.Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.

Measurement of IL-21 in human serum and plasma using ultrasensitive MSD S-PLEX® and Quanterix SiMoA methodologies.

IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format. Assays developed on both technology platforms were characterized in heparin plasma and serum using spike recoveries across a range of concentrations. Each method was able to detect sub-pg/mL levels of IL-21 (predicted Limit of Detection [LOD] of approximately 1.0 fg/mL for both the Quanterix SiMoA and MSD S-PLEX® platforms) which is 200-500 times lower than current commercial assays. Additionally we demonstrated that rheumatoid factor did not interfere with measuring IL-21 in the Quanterix SiMoA assay. Results obtained with the two new ultrasensitive assays showed a strong correlation (r = 0.9428; p < .0001). Additionally, IL-21 levels were significantly increased in samples from patients with Systemic Lupus Erythematosus (mean+/- SD: n = 14, 202.64 +/- 111.47 fg/mL, p = .0001 for Quanterix SiMoA and 275.4 +/- 174.66 fg/mL p = .0001 for MSD S-PLEX®) as well as in samples from patients with Sjögren's Syndrome (mean+/- SD: n = 11, 122.18 +/- 84.50 fg/mL, p = .0029 for Quanterix SiMoA and 183.64 +/- 153.00 fg/mL, p = .0082 for MSD S-PLEX®) when compared to healthy donors (mean+/- SD: n = 11, 38.1 +/- 27.8 fg/mL for Quanterix SiMoA and 58.1 +/- 30.7 fg/mL for MSD S-PLEX®). These ultrasensitive assays, for the first time, allow for the accurate quantitation of human IL-21 in heparin plasma and serum. In addition, these experiments also provide a direct comparison of the MSD S-PLEX® format and Quanterix SiMoA platform technologies, which may have broader implications to future application of these methods to evaluate low abundance proteins in complex biological matrices.

Effect of mite allergenic components on innate immune response: Synergy of protease (Group 1 & 3) and non-protease (Group 2 & 7) allergens.

The major mite allergenic components of protease allergens (group 1,3) and non-protease allergens (group 2,7) derived from Dermatophagoides peronyssinus (Dp) and D. farinae (Df) are reported to be capable of sensitizing 80-90% of mite-allergic patients. Although protease and non-protease allergens have been demonstrated to trigger innate and adaptive immune responses through epithelium activation, the simultaneous or sequential effects of both groups of allergens has not been reported. Since all allergens are present in the mite crude extracts, it is important to determine whether these allergens can synergistically trigger the immune responses to cause airway inflammation. A total of 60 house dust mite (HDM)-allergic asthmatic patients were recruited to analyze their serum-specific IgE response to both groups of allergens. Recombinant protease allergen (Der p1 and Der p3) and non-protease allergens (Der p2 and Der p7) were used to activate the human airway epithelium cell (Beas-2B). The cells were analyzed for mRNA expression of IL-6/IL-8 and the culture supernatants were analyzed for neutrophil chemotactic activity (NCA). The results showed 48/60 (80%) HDM-allergic patients were sensitized to all allergenic components of Der p1, Der p2, Der f1, and Der f2. Most of the allergic patients were sensitized to both groups of allergens simultaneously. The associations of Der p1 with Der p2 were 83.3% (50/60) and Der f1 with Der f2 were 80% (48/60). When Beas-2B cells were cultured with Der p2 in conjunction with Der p1 and Der p3, the results showed that there was increased expression of IL-6/IL-8 in comparison with culture with allergen alone. There was only a trivial effect on IL-6/IL-8 expression when Der p2 was co-cultured with Der p7. Similar findings were obtained in the NCA measurement. When Beas-2B was cultured with Der p2 in conjunction with Der p1 and Der p3, there was increased NCA in comparison with culture with allergen alone. There were also trivial effects when Der p2 was co-cultured with Der p7. The allergens (Der p2 and Der p3)-induced IL-6/IL-8 expression and NCA released from Beas-2B could be downregulated by dexamethasone and transcription factor inhibitor SP600125. The allergenic components derived from Dp and Df can sensitize allergic patients simultaneously and activate epithelium through protease allergens (group 1, 3) and non-protease allergen (group 2) synergistically.

Comparison of Allergenicity at Gly m 4 and Gly m Bd 30K of Soybean after Genetic Modification.

Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

Reversible Single-Crystal to Single-Crystal Transformations of a Zn(II)-Salicyaldimine Coordination Polymer Accompanying Changes in Coordination Sphere and Network Dimensionality upon Dehydration and Rehydration.

A fluorescent Zn(II)-salicyaldimine coordination polymer, [Zn(L(salpyca))(H2O)]n (1; H2L(salpyca) = 4-hydroxy-3-(((pyridin-2-yl)methylimino)methyl)benzoic acid), showing a one-dimensional (1D) zigzag chain structure has been hydro(solvo)thermally synthesized. Removal of coordination water molecules in 1 by thermal dehydration gives rise to the dehydration product [Zn(L(salpyca))]n (1'), which has a dizinc-based two-dimensional (2D) gridlike (4,4)-layer structure. X-ray powder diffraction (XRPD) patterns, thermogravimetric (TG) analyses, and infrared (IR) spectra all clearly indicate that the structure of 1 is quite flexible as a result of a reversible 1D-2D single-crystal to single-crystal (SCSC) transformation upon removal and rebinding of coordination water molecules, which accompanies changes in coordination sphere and network dimensionality. Additionally, Zn(II)-salicyaldimine polymers 1 and 1' exhibit different solid-state photoluminescences at 458 and 480 nm, respectively. This is reasonably attributed to the close-packing effect and/or the influences of the differences on the conformation and the coordination mode of the L(salpyca) ligand and the coordination geometry around the Zn(II) center.

Prediction of colorectal cancer diagnosis based on circulating plasma proteins.

Non-invasive detection of colorectal cancer with blood-based markers is a critical clinical need. Here we describe a phased mass spectrometry-based approach for the discovery, screening, and validation of circulating protein biomarkers with diagnostic value. Initially, we profiled human primary tumor tissue epithelia and characterized about 300 secreted and cell surface candidate glycoproteins. These candidates were then screened in patient systemic circulation to identify detectable candidates in blood plasma. An 88-plex targeting method was established to systematically monitor these proteins in two large and independent cohorts of plasma samples, which generated quantitative clinical datasets at an unprecedented scale. The data were deployed to develop and evaluate a five-protein biomarker signature for colorectal cancer detection.

Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter.

PURPOSE: Myeloid differentiation-2 (MD-2) has been associated with endotoxin and inflammatory disorders because it can recognize lipopolysaccharide (LPS) binding and attenuate Toll-like receptor 4 (TLR4)-mediated signaling. However, its role in allergic inflammation has yet to be clarified. We examined whether single nucleotide polymorphisms (SNPs) in MD-2 promoter can affect MD-2 expression and aimed to clarify the relationship between Der p 2 allergy and SNPs of MD-2 promoter. METHODS: The function of SNPs of MD-2 promoter and the effects of cytokines and immunoglobulin on the secretion and mRNA expression were investigated in 73 allergic subjects with different MD-2 gene promoter variants. Peripheral blood mononuclear cells were cultured with or without LPS in the presence of Dermatophagoides pteronyssinus group 2 allergen (Der p 2), followed by mRNA extraction and cytokine expression analysis. The culture supernatants were collected for cytokine measurement. RESULTS: Patients with the MD-2 promoter SNPs (rs1809441/rs1809442) had increased mRNA expressions of MD-2, ε heavy chain of IgE (Cε), and interleukin (IL)-8; however, only MD-2 and IL-8 were further up-regulated after Der p 2 stimulation. Patients with SNPs of MD-2 promoter tended to have high levels of IL-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α after Der p 2 and LPS stimulation. Increased secretions of IL-6, IL-8, and IL-10 were found to be up-regulated by Der p 2 stimulation, and an increased secretion of IFN-γ and decreased secretion of IL-4 were noted after LPS stimulation. CONCLUSIONS: The high levels of proinflammatory cytokines secreted by Der p 2 were predetermined by MD-2 promoter SNPs (rs1809441/rs1809442). Through cytokine secretion by Der p 2 and LPS, these SNPs may serve as an indicator of the pathological phenotype of Der p 2-induced allergic inflammation.

An Exploratory Pilot Study of Genetic Marker for IgE-Mediated Allergic Diseases with Expressions of FcεR1α and Cε.

The high affinity immunoglobulin E (IgE) receptor-FcεR1 is mainly expressed on the surface of effector cells. Cross-linking of IgE Abs bound to FcεR1 by multi-valent antigens can induce the activation of these cells and the secretion of inflammatory mediators. Since FcεR1 plays a central role in the induction and maintenance of allergic responses, this study aimed to investigate the association of FcεR1 with the allergic phenotype of Cε expression and cytokine and histamine release from peripheral leukocytes. Peripheral leukocytes from 67 allergic and 50 non-allergic subjects were used for genotyping analysis. Peripheral mononuclear cells (PBMCs) were used for Cε expression and ELISpot analysis, while polymorphonuclear cells (PMNs) were used for histamine release. The association between genotype polymorphism of the FcεR1α promoter region (rs2427827 and rs2251746) and allergic features of Cε expression and histamine were analyzed, and their effects on leukocytes function were compared with wild type. The genotype polymorphisms of FcεR1α promoter region with CT and TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with single nucleotide polymorphism (SNP) of FcεR1α promoter region had high levels of total IgE, mite-specific Der p 2 (Group 2 allergen of Dermatophagoides pteronyssinus)-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with SNPs of the FcεR1α promoter region. Despite the increased Cε mRNA expression in PBMCs and histamine release from PMNs and the up-regulated mRNA expression of interleukin (IL)-6 and IL-8 secretions after Der p2 stimulation, there was no statistically significant difference between SNPs of the FcεR1α promoter region and the wild type. SNPs of FcεR1α promoter region were associated with IgE expression, IgE producing B cells, and increased Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by Dermatophagoides pteronyssinus.

Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling.

PURPOSE: House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation. METHODS: Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion. CONCLUSIONS: Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.

Association of Single Nucleotide Polymorphisms in the MD-2 Gene Promoter Region With Der p 2 Allergy.

PURPOSE: Sensitization to house dust mite (Dermatophagoides pteronyssinus) is a considerable risk factor for the progression of allergic disease. The group 2 allergen from Dermatophagoides pteronyssinus, Der p 2, is considered a major one in patients with specific immunoglobulin E (IgE) to Der p 2. Der p 2 has structural homology with myeloid differentiation 2 (MD-2), which is involved in the lipopolysaccharide-binding component of the Toll-like receptor 4 signaling pathway and the development of inflammation. The aim of this study was to examine the genetic association of single nucleotide polymorphisms (SNPs) in the promoter region of MD-2 with Der p 2-sensitive allergy. METHODS: We investigated associations between cohort's characteristics, including 280 allergic and 80 healthy subjects by examining total IgE, eosinophils, D. pteronyssinus-specific IgE, Der p 2-specific IgE, the number of IgE-producing B cells induced by Der p 2, and the odds ratio of allergic symptoms. RESULTS: Based on the 1,000 genome project data, the minor allele frequencies of the rs1809441 and rs1809442 are 0.467 and 0.474, respectively. However, the correlation of linkage disequilibrium (LD) between these 2 SNPs is D'=1, the genotype frequencies of the 2 MD-2 (LY96) SNPs (rs1809441 and rs1809442) that are located nearby were significantly different between allergic and health subjects: the TT genotype of rs1809441 and the GG genotype of rs1809442 were more frequent in allergic subjects than in healthy subjects (16.1% vs 2.5% in both genotypes). The allergic patients with these genotypes exhibited significantly higher levels of D. pteronyssinus-specific IgE and Der p 2-specific Ig E, and a larger number of Der p 2-activated B cells. In addition, these 2 SNPs in the MD-2 promoter region were significantly associated with the prevalence of nasal, skin, and asthmatic allergic symptoms. CONCLUSIONS: Our results indicated that 2 SNPs in the MD-2 promoter region were significantly associated with Der p 2-specific Ig E, and thereby suggest that these SNPs may play a major role in susceptibility to Der p 2-triggered immune responses in a Taiwanese population.