Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Background: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 mu g and less) and has a sound reproducibility. Conclusions: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner

Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice

Introduction: Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. Methods: To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. Results: In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXR and LXR isoforms (LXR-/--/-) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXR-/--/- mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4(+) lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. Conclusions: In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production

Angiogenin released from ABCB5+ stromal precursors improves healing of diabetic wounds by promoting angiogenesis

Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors–based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis

Newly defined ATP-binding cassette subfamily B member 5 positive dermal mesenchymal stem cells promote healing of chronic iron-overload wounds via secretion of interleukin-1 receptor antagonist

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP‐binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+‐derived mesenchymal stem cells (MSCs) attenuated macrophage‐dominated inflammation and thereby accelerated healing of full‐thickness excisional wounds in the iron‐overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin‐1 receptor antagonist (IL‐1RA) secreted by ABCB5+‐derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti‐inflammatory M2 macrophages at the wound site. The beneficial anti‐inflammatory effect of IL‐1RA released from ABCB5+‐derived MSCs on human wound macrophages was conserved in humanized NOD‐scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker‐enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans

Mycobacterium tuberculosis bio-lipid mycolic acid and mammalian cholesterol-sensor liver X receptor in airway immunity

Within this dissertation we have first addressed macrophage (MΦ) and airway responses in mice to the complex Mycobacterium tuberculosis bio-lipid mycolic acid (MA) with respect to its chemical fine-structure. This revealed a distinct inflammatory capacity, and even anti-inflammatory effects, dependent on the MA cyclopropane stereochemistry and oxygenation type. These chemical moieties on MAs also impacted on the induction of foam cells due to MΦ intracellular cholesterol accumulation. Also, the MA chemistry was found to be important for the previously described T-helper 2 cell (Th2)-tolerizing anti-inflammatory effect in the airways of asthma model mice, coinciding with shifts in Th-cell lineages balances. Additionally, we have identified possible novel molecular interactions between MA and host cell signaling protein molecules that could in part explain the observed biological functions. Thus, MAs could selectively precipitate the 65kDA regulatory subunit of protein phosphatase A from mouse MΦ cytosolic protein lysates and they could furthermore suppress the ligand-mediated activation of gene-transcription by retinoid X receptor-permissive nuclear receptors, liver X receptor (LXR) and retinoic acid receptor. All three of these mammalian proteins are involved in cellular lipid household and lipid signaling events, but also exert some level of control on inflammation and immunity. Second, the importance of LXR subtypes α and β for protection against pulmonary M. tuberculosis infection was studied and we found that functional LXR-α in particular was required for full-blown neutrophil and Th17-mediated anti-tuberculous immunity. Further dissection of this observed immune control by LXR was performed based on the application of an in-house established non-infectious Th1/Th17-driven airway allergic inflammatory model for neutrophilic severe refractory asthma. This revealed that LXR-α knock-out (KO) and LXR-α,β double KO mice, but not LXR-β KO or wild-type mice, indeed mirrored the Th17 immune defect observed in the infectious model. This was further ascribed to the lack of LXR endogenous functions at the time of allergic inflammation in response to airway allergen exposure, rather than to Th-cell intrinsic effects. In contrast, by the use of an analogous mouse model for mild eosinophilic asthma, we observed that the Th2-mediated eosinophil inflammatory response was reduced by LXR-deficiency. Preliminary data identify possible functional involvement of LXRs in processes driving eosinophil chemotaxis to the site of inflammation and Th2-cel cytokine-induced de novo eosinopoiesis by bone marrow progenitor cells in response to repeated airway allergen exposure. In conclusion, the data presented here are indicative for an active utilization of the host lipid signaling pathways by mycobacterial MAs to manipulate the host cellular response to infection. This level of control could further be fine-tuned by the relative abundance of certain types of MA chemical modifications and that were here associated with distinct potencies of MAs to exert biological functions in inflammation and cholesterol homeostasis. Additionally, we have shown that the mammalian cholesterol homeostasis regulator LXR is indispensible for effective anti-tuberculous Th17-dependent immunity and furthermore contributes to allergen-induced Th2-effector eosinophil responsivity in the airways

Photodynamic Therapy, Photobiomodulation and Acetonide Triamcinolone 0.1% in the Treatment of Oral Lichen Planus: A Randomized Clinical Trial

Objective: To evaluate the efficacy of photodynamic therapy (PDT) and photobiomodulation (PBM) in the treatment of oral lichen planus (OLP) in comparison with the use of topical corticosteroids. Material and methods: Sixty patients with OLP were randomized to three groups: group 1 photodynamic therapy applied once a week for four sessions, with orabase cream; group 2 low-power laser application with orabase cream; and group 3 inactive laser with triamcinolone acetonide 0.1%. Patient pain was evaluated, and the Thongprasom severity score, the Oral Health Impact Profile-14 (OHIP-14), and the Hamilton anxiety and depression scale at one and three months of follow-up. (ClinicalTrials.gov Identifier: NCT05127083). Results: Pain decreased significantly over time in all groups, though the symptoms relapsed over follow-up at one and three months in group 3. The OHIP-14 score improved significantly in groups 1 and 2 (p p = 0.032) and between groups 2 and 3 (p = 0.024). Conclusions: Photodynamic therapy and photobiomodulation once a week for four weeks are safe and non-invasive treatment options, with the important advantage of lacking adverse effects. Further studies are needed to confirm it

Innate imprinting of murine resident alveolar macrophages by allergic bronchial inflammation causes a switch from hypoinflammatory to hyperinflammatory reactivity

Resident alveolar macrophages (rAMs) residing in the bronchoalveolar lumen of the airways play an important role in limiting excessive inflammatory responses in the respiratory tract High phagocytic activity along with hyporesponsiveness to inflammatory insults and lack of autonomous IFN-beta production are crucial assets in this regulatory function. Using a mouse model of asthma, we analyzed the fate of rAMs both during and after allergic bronchial inflammation. Although nearly indistinguishable phenotypically from naive rAMs, postinflammation rAMs exhibited a strongly reduced basal phagocytic capacity, accompanied by a markedly increased inflammatory reactivity to Toll-like receptors TLR-3 (poly I:C), TLR-4 [lipopolysaccharide (LPS)], and TLR-7 (imiquimod). Importantly, after inflammation, rAMs exhibited a switch from an IFN-beta-defective to an IFN-beta-competent phenotype, thus indicating the occurrence of a new, inflammatory-released rAM population in the postallergic lung. Analysis of rAM turnover revealed a rapid disappearance of naive rAMs after the onset of inflammation. This inflammation-induced rAM turnover is critical for the development of the hyperinflammatory rAM phenotype observed after clearance of bronchial inflammation. These data document a novel mechanism of innate imprinting in which noninfectious bronchial inflammation causes alveolar macrophages to acquire a highly modified innate reactivity. The resulting increase in secretion of inflammatory mediators on TLR stimulation implies a role for this phenomenon of innate imprinting in the increased sensitivity of postallergic lungs to inflammatory insults. (Am J Pathol 2012, 181:174-184; http://dx.doi.org/10.1016/j.ajpath.2012.03.015

Liver X receptors contribute to the protective immune response against Mycobacterium tuberculosis in mice

Liver X receptors (LXRs) are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. However, exactly how LXRs modulate inflammation during infection remains unknown. To explore this, we used a mouse model of Mycobacterium tuberculosis infection. Upon intratracheal infection with M. tuberculosis, LXRs and LXR target genes were induced in CD11c+ lung and alveolar cells. Furthermore, mice deficient in both LXR isoforms, LXRα and LXRβ (Lxra–/–Lxrb–/– mice), were more susceptible to infection, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. Interestingly, mice solely deficient in LXRα, but not those lacking only LXRβ, mirrored the susceptibility of the Lxra–/–Lxrb–/– animals. Lxra–/–Lxrb–/– mice failed to mount an effective early neutrophilic airway response to infection and showed dysregulation of both pro- and antiinflammatory factors in CD11c+ lung cells. T cell responses were strongly affected in Lxra–/–Lxrb–/– mice, showing near-complete abrogation of the infection-induced Th1 function — and even more so Th17 function — in the lungs. Treatment of WT mice with the LXR agonists TO901317 and GW3965 resulted in a 10-fold decrease of the pulmonary bacterial burden and a comparable increase of Th1/Th17 function in the lungs. The dependence of LXR signaling on the neutrophil IL-17 axis represents what we believe to be a novel function for these nuclear receptors in resistance to M. tuberculosis infection and may provide a new target for therapeutics