Induction Of Lesions Research Articles

027 Memory B cells of atopic individuals preferentially express IL-31RA: a putative role of IL-31 in B cell biology

The novel cytokine IL-31 and its receptor have been shown to play a central role in bridging the immune system with neurons, epithelial surfaces and connective tissues. Although increasing evidence has demonstrated their role in inflammation, their involvement in the expansion of distinct leukocyte subsets is not fully understood. Detailed analyses of Il31-transgenic (Eμ-Lck) mice indicated that next to the induction of pruritus and skin lesions, mice also presented enlarged lymph nodes with increased B cell frequency. Therefore, we aimed at characterizing the role of IL-31 signaling in B cell biology. Using flow cytometry, the frequency of total B cells and specific B cell subsets was assessed in peripheral lymph nodes and bone marrow of Il31-transgenic (Eμ-Lck) mice and wild-type controls. Moreover, IL-31RA expression was analyzed on human B cell subsets from peripheral blood of healthy and atopic dermatitis donors. We observed a significantly increased frequency of CD19+ B cells in bone marrow and peripheral lymph nodes in Il31-transgenic mice. Detailed analyses of B cell subsets indicated that Il31-transgenic mice showed a higher frequency of plasmablasts in peripheral lymph nodes. In human, memory B cells represented the major population expressing IL-31RA within the B cell compartment. Furthermore, atopic dermatitis patients with elevated IgE levels (>1000 kU/l) showed a higher IL-31RA expression on memory B cells compared to healthy donors. This is of particular interest since DOCK8 acts as a negative regulator of IL-31. DOCK8 deficiency-related hyper-IgE syndrome presents with elevated serum IgE levels and severe atopic dermatitis with increased IL-31 expression. Taken together, these findings point to a novel role of IL-31 in B cell biology and an atopic IgE-producing phenotype.

GPER1 links estrogens to centrosome amplification and chromosomal instability in human colon cells.

The role of the alternate G protein-coupled estrogen receptor 1 (GPER1) in colorectal cancer (CRC) development and progression is unclear, not least because of conflicting clinical and experimental evidence for pro- and anti-tumorigenic activities. Here, we show that low concentrations of the estrogenic GPER1 ligands, 17β-estradiol, bisphenol A, and diethylstilbestrol cause the generation of lagging chromosomes in normal colon and CRC cell lines, which manifest in whole chromosomal instability and aneuploidy. Mechanistically, (xeno)estrogens triggered centrosome amplification by inducing centriole overduplication that leads to transient multipolar mitotic spindles, chromosome alignment defects, and mitotic laggards. Remarkably, we could demonstrate a significant role of estrogen-activated GPER1 in centrosome amplification and increased karyotype variability. Indeed, both gene-specific knockdown and inhibition of GPER1 effectively restored normal centrosome numbers and karyotype stability in cells exposed to 17β-estradiol, bisphenol A, or diethylstilbestrol. Thus, our results reveal a novel link between estrogen-activated GPER1 and the induction of key CRC-prone lesions, supporting a pivotal role of the alternate estrogen receptor in colon neoplastic transformation and tumor progression.

Selenium ameliorates inflammation by decreasing autophagic flux and mitogen-activated protein kinase signalling on experimentally induced rat periapical lesions.

To reveal the molecular mechanisms that targets mitogen-activated protein kinase (MAPK) signalling and the autophagic flux and to investigate the possible effects of the systemic administration of selenium (Se) on experimentally induced rat periapical lesions. Thirty adult Sprague-Dawley rats were divided equally into negative control, positive control and Se groups. In the positive control and Se groups, the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions The Se group received daily intraperitoneal injections of Se at a dose of 0.1mg kg-1 . After 28 days, the amount of bone destruction; severity of inflammation; penetration of microorganisms along the root canal; collagen degradation in periodontal ligament; interleukin (IL)-6, hypoxia-inducible factor-1 (HIF-1), cyclooxygenase-2 (COX-2) and caspase-3 expression; autophagic flux; and p38 MAPK signalling were evaluated using radiographic, histopathological, Gram staining, picrosirius red stain, immunohistochemical, quantitative real-time polymerase chain (qRT-PCR) and Western blot methods, respectively. These data were analysed through the Kruskal-Wallis and Dunnett's tests (p < .05). The area of radiographic periapical bone loss, histopathological scores, the area of periapical bone loss and the scores for the bacteria localisation, the intensity of immunohistochemical staining for IL-6, HIF-1, COX-2 and caspase-3 in the Se group was significantly less than those of the positive control group (p < .01). The mRNA expression levels of Beclin-1, Atg3, Atg5, Atg7 and Atg16L1 were lower in the Se group than in the positive control group (p < .01). The protein expressions of Beclin-1, Atg5 and LC3-II, the phosphorylation ratio of the p38 MAPK and the ratios of LC3II/LC3I were significantly higher (p < .05) in the positive control and Se groups. On the contrary, the expression of the p62/SQSTM1 protein was significantly lower (p < .05) in the positive control and Se groups than in the negative control group. The induction of periapical lesions in rats increased autophagic flux and activated p38 MAPK signal transduction processes. Se suppressed the inflammatory process, reduced bone destruction and both the autophagic flux and p38 MAPK activation.

Modelling the HPRT-gene mutation induction of particle beams: systematic in vitro data collection, analysis and microdosimetric kinetic model implementation

Objective. Since the early years, particle therapy treatments have been associated with concerns for late toxicities, especially secondary cancer risk (SCR). Nowadays, this concern is related to patients for whom long-term survival is expected (e.g. breast cancer, lymphoma, paediatrics). In the aim to contribute to this research, we present a dedicated statistical and modelling analysis aiming at improving our understanding of the RBE for mutation induction () for different particle species. Approach. We built a new database based on a systematic collection of RBE data for mutation assays of the gene encoding for the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase from literature (105 entries, distributed among 3 cell lines and 16 particle species). The data were employed to perform statistical and modelling analysis. For the latter, we adapted the microdosimetric kinetic model (MKM) to describe the mutagenesis in analogy to lethal lesion induction. Main results. Correlation analysis between RBE for survival (RBES) and reveals significant correlation between these two quantities (ρ = 0.86, p < 0.05). The correlation gets stronger when looking at subsets of data based on cell line and particle species. We also show that the MKM can be successfully employed to describe obtaining comparably good agreement with the experimental data. Remarkably, to improve the agreement with experimental data the MKM requires, consistently in all the analysed cases, a reduced domain size for the description of mutation induction compared to that adopted for survival. Significance. We were able to show that RBES and are strongly related quantities. We also showed for the first time that the MKM could be successfully applied to the description of mutation induction, representing an endpoint different from the more traditional cell killing. In analogy to the RBES, can be implemented into treatment planning system evaluations.

Distribution and efficacy of ofatumumab and ocrelizumab in humanized CD20 mice following subcutaneous or intravenous administration.

Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain.

LB868 Th2 skewing promotes the expression of skin-homing molecules on T cells and is required for the induction of skin lesions in lupus-prone mice

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by high IFN expression. Recent advances have challenged the Th1-Th2 paradigm in CLE, as there are reports of expression of both Th1 and Th2 cytokines in lesional skin. We assessed the role of T cell skewing in the initiation of skin disease using a lupus-prone mouse model of CLE that depends on recognizing cognate antigen by infiltrating T cells in an immune environment perturbed by a TLR7-driven type I IFN response.

Vascular calcification in different arterial beds in ex vivo ring culture and in vivo rat model

Vascular calcification is a risk factor for cardiovascular and kidney diseases. Medial calcification may differently affect the arterial tree depending on vessel location and smooth muscle injury. The aim was to map the anatomical distribution of vascular calcifications on different arteries and artery locations, in cultured artery rings (ex vivo) and in a rat model of elastocalcinosis (in vivo). Vascular calcification was assessed histologically (von Kossa staining of the media) and by calcium content measurement. Arteries of different sizes were harvested from untreated rats for ring culture and from the vitamin D3-nicotine (VDN) rat model for direct observation. When cultured in pro-calcifying conditions, thoracic aorta exhibited similar calcification from the arch to the diaphragm. Calcification increased in abdominal aorta along with the reduction in cross sectional area. Carotid and renal arteries exhibited similar ex vivo calcification. In VDN rats, calcification was greater in carotid artery than in aorta, and was accompanied by fibrosis and apoptosis. Ex vivo, calcification was increased by the induction of lesions on arteries. Along the vascular tree, calcification of the arterial wall increases with the narrowing of vessels in ex vivo ring culture and in vivo. The observed differences represent local susceptibility of the vessels to the calcifying processes.

Pharmacological inactivation of the primate posterior insular/secondary somatosensory cortices attenuates thermal hyperalgesia.

We previously established a macaque model of central post-stroke pain (CPSP) and confirmed the involvement of increased activity of the posterior insular cortex (PIC) and secondary somatosensory cortex (SII) to somatosensory stimuli in mechanical allodynia by a combination of imaging techniques with local pharmacological inactivation. However, it is unclear whether the same intervention would be effective for thermal hyperalgesia. Therefore, using the macaque model, we examined behavioural responses to thermal stimuli following pharmacological inactivation of the PIC/SII. Two CPSP model macaques were established based on collagenase-induced unilateral hemorrhagic lesions in the ventral posterolateral nucleus of the thalamus. To evaluate pain perception, withdrawal latencies to thermal stimuli of 37, 45, 50, 52, and 55 °C to hands were measured. Several weeks after the lesion induction, pharmacological inactivation of the PIC/SII by microinjection of muscimol was performed. The effect of inactivation on withdrawal latency was assessed by comparison with withdrawal latency after vehicle injection. Several weeks after induction of the thalamic lesions, both macaques demonstrated a reduction in withdrawal latencies to thermal stimulation (<50 °C) on the contralesional hand, indicating the occurrence of thermal hyperalgesia. When the PIC/SII were inactivated by muscimol, the withdrawal latencies to thermal stimuli of 50 and 52 °C were significantly increased compared to those after vehicle injection. Our data emphasize that increased activity in the PIC/SII after appearance of thalamic lesions can contribute to abnormal pain of multiple modalities, and the modulation of PIC/SII activity may be a therapeutic approach for thermal hyperalgesia. CPSP is caused by stroke lesions in the sensory system and characterized by mechanical allodynia or thermal hyperalgesia. Inactivation of the PIC/SII has an analgesic effect on mechanical allodynia; however, it is not clear whether the same intervention could reduce thermal hyperalgesia. Here, using the macaque model, we demonstrated that inactivation of these cortices reduces hypersensitivity to thermal stimuli. This result emphasizes that increased PIC/SII activity can contribute to abnormal pain of multiple modalities.

Th2 to Th1 Transition Is Required for Induction of Skin Lesions in an Inducible and Recurrent Murine Model of Cutaneous Lupus-Like Inflammation.

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by a strong IFN signature, normally associated with type I IFNs. However, increasing evidence points to an additional role for IFNγ, or at least a pathogenic T effector subset dependent on IFNγ, for disease progression. Nevertheless, Th2 effector subsets have also been implicated in CLE. We have now assessed the role of specific T cell subsets in the initiation and persistence of skin disease using a T cell-inducible murine model of CLE, dependent on KJ1-26 T cell recognition of an ovalbumin fusion protein. We found that only Th2-skewed cells, and not Th1-skewed cells, induced the development of skin lesions. However, we provide strong evidence that the Th2 disease-initiating cells convert to a more Th1-like functional phenotype in vivo by the time the skin lesions are apparent. This phenotype is maintained and potentiates over time, as T cells isolated from the skin, following a second induction of self-antigen, expressed more IFN-γ than T cells isolated at the time of the initial response. Transcriptional analysis identified additional changes in the KJ1-26 T cells at four weeks post injection, with higher expression levels of interferon stimulated genes (ISGs) including CXCL9, IRF5, IFIH1, and MX1. Further, injection of IFN-γ-/- T cells faied to induce skin disease in mice. We concluded that Th2 cells trigger skin lesion formation in CLE, and these cells switch to a Th1-like phenotype in the context of a TLR7-driven immune environment that is stable within the T cell memory compartment.

An experimental model to induce digital dermatitis in beef calves

BackgroundDigital dermatitis (DD) is a multifactorial infectious disease affecting the skin on feet of cattle causing erosion and inflammation above the heel bulbs. Some cases of DD cause lameness and significantly impact animal welfare and productivity. While DD has emerged as a concern for the beef industry, key information regarding early detection and its impact on cattle behaviour is lacking. The primary objective of this study was to determine if an established DD experimental model for dairy calves could be used to induce DD lesions in beef calves. A secondary objective was to describe changes in behaviour and pain associated with induction of DD lesions. Eight beef calves acquired from a single cow-calf operator were enrolled in the study. Upon enrolment, calves were evaluated and determined to be free of foot lesions. Within the experimental environment, calves were housed in individual pens and assigned to two groups (mock-inoculated and inoculated). Both hind feet of each calf were enrolled. Within calf, inoculation protocol was consistent, and a 28-day experimental protocol was employed. Two days prior to inoculation, both hind feet of each calf were abraded (area above the heel bulbs and below the dewclaws), moistened, and wrapped to facilitate an anaerobic condition. Feet were inoculated with macerated DD lesion material or mock inoculum and remained wrapped until clinical signs of DD or protocol endpoint.ResultsAfter a period of 14 to 18 days post inoculation, three of five inoculated calves developed clinical signs (lameness), and upon close inspection, DD lesions were present on at least one hind foot. Two of five inoculated calves did not develop lesions within 28 days. Zero of three mock-inoculated calves developed DD. Treponema spp. were detected by quantitative polymerase chain reaction from biopsies of induced lesions. Measurements of behaviour prior to disease induction were numerically different between DD affected and mock-inoculated calves.ConclusionsAn experimental infection model established for dairy cattle was used to successfully induce acute DD lesions in three of five inoculated beef calves. This model can provide a framework to study intervention protocols and to evaluate the impact of DD on behaviour and pain.

Rho-Kinase Inhibition Improves the Outcome of Focal Subcortical White Matter Lesions.

Subcortical white matter lesions are exceedingly common in cerebral small vessel disease and lead to significant cumulative disability without an available treatment. Here, we tested a rho-kinase inhibitor on functional recovery after focal white matter injury. A focal corpus callosum lesion was induced by stereotactic injection of N5-(1-iminoethyl)-L-ornithine in mice. Fasudil (10 mg/kg) or vehicle was administered daily for 2 weeks, starting one day after lesion induction. Resting-state functional connectivity and grid walk performance were studied longitudinally, and lesion volumes were determined at one month. Resting-state interhemispheric functional connectivity significantly recovered between days 1 and 14 in the fasudil group (P<0.001), despite worse initial connectivity loss than vehicle before treatment onset. Grid walk test revealed an increased number of foot faults in the vehicle group compared with baseline, which persisted for at least 4 weeks. In contrast, the fasudil arm did not show an increase in foot faults and had smaller lesions at 4 weeks. Immunohistochemical examination of reactive astrocytosis, synaptic density, and mature oligodendrocytes did not reveal a significant difference between treatment arms. These data show that delayed fasudil posttreatment improves functional outcomes after a focal subcortical white matter lesion in mice. Future work will aim to elucidate the mechanisms.

Time course of dorsolateral geniculate nucleus plasticity in adult monkeys with laser-induced retinal lesions.

We studied changes in the expression of growth-associated protein 43 (GAP43), glial fibrillary acidic protein (GFAP), and calcium-binding proteins (calbindin [Cb] and parvalbumin [Pv]) in the dorsal lateral geniculate nucleus (dLGN) of four capuchin monkeys with laser-induced retinal lesions. The lesions were generated with the aid of a neodymium-YAG dual-frequency laser with shots of different intensity and at different survival time in each animal. The expression of these proteins in the layers of the dLGN was evaluated by performing histodensitometry of coronal sections throughout the nucleus. High-power laser shots administered at the border of the optic disc (OD)-injured fibers resulted in large scotomas. These lesions produced a devastating effect on fibers in this passage, resulting in large deafferentation of the dLGN. The time course of plasticity expressed in this nucleus varied with the degree of the retinal lesion. Topographically, corresponding portions of the dLGN were inferred by the extent of the ocular dominance column revealed by cytochrome oxidase histochemistry in flattened preparations of V1. In the region representing the retinal lesion, the expression of GFAP, GAP43, Pv, and Cb increased and decreased in the corresponding dLGN layers shortly after lesion induction and returned to their original values with different time courses. Synaptogenesis (indicated by GAP43 expression) appeared to be increased in all layers, while "cleansing" of the glial-damaged region (indicated by GFAP expression) was markedly greater in the parvocellular layers, followed by the magnocellular layers. Schematic drawings of optic discs laser lesions and of series of coronal sections of the dLGN, in three monkeys, depicting the areas of the nucleus deafferented by the lesions.

A Mouse Model of Endometriosis with Nanoparticle Labeling for In Vivo Photoacoustic Imaging.

Endometriosis is a condition of the female reproductive tract characterized by endometrium-like tissue growing outside the uterus. Though it is a common cause of pelvic pain and infertility, there is currently no reliable noninvasive method to diagnose the presence of endometriosis without surgery, and the pathophysiological mechanisms that lead to the occurrence of symptoms require further inquiry. Due to patient heterogeneity and delayed diagnosis, animal models are commonly used to study the development of endometriosis, but these are costly due to the large number of animals needed to test various treatments and experimental conditions at multiple endpoints. Here, we describe a method for synthesis of multimodal imaging gold-fluorescein isothiocyanate (FITC) nanoparticles with preclinical application via induction of nanoparticle-labeled endometriosis-like lesions in mice. Labeling donor endometrial tissue fragments with gold-FITC nanoparticles prior to induction of endometriosis in recipients enables in vivo detection of the gold-labeled lesions with photoacoustic imaging. The same imaging method can be used to visualize embryos noninvasively in pregnant mice. Furthermore, the conjugated FITC dye on the gold nanoparticles allows easy isolation of labeled lesion tissue under a fluorescence dissection microscope. After dissection, the presence of gold-FITC nanoparticles and endometrium-like histology of lesions can be verified through fluorescence imaging, gold enhancement, and immunostaining. This method for in vivo imaging of endometriosis-like lesions and fluorescence-guided dissection will permit new experimental possibilities for the longitudinal study of endometriosis development and progression as well as endometriosis-related infertility.

An Experimental Dermal Oedema Model for Apx Toxins of Actinobacillus pleuropneumoniae

In-vivo models of Actinobacillus pleuropneumoniae (App) infection in pigs are required for the development of vaccines and investigations of pathogenicity. Existing models cause severe respiratory disease with pulmonary oedema, dyspnoea and severe thoracic pain, and careful monitoring and early intervention with euthanasia is, therefore, needed to avoid unnecessary suffering in experimental animals. As a potential replacement for the existing respiratory infection model, an in-vivo protocol was evaluated using intradermal or subcutaneous injection of different App strains and Apx toxins into the abdominal skin of pigs. High concentrations of serovar 1 and serovar 10 App induced diffuse visible dermal oedema and inflammation. Injection of Apx toxins alone did not adequately produce macroscopic lesions, although an influx of inflammatory cells was seen on histopathology. ApxI-producing strains of App induced more inflammation than ApxII- and ApxIII-producing strains. Induction of skin lesions by injection of App or Apx toxins was not sufficiently repeatable or discrete for a robust experimental model that could be used for assessment of novel interventions.

LC-MS/MS for Assessing the Incorporation and Repair of N2-Alkyl-2'-deoxyguanosine in Genomic DNA.

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N2-MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N2-alkyl-dG lesions in mammalian cells.

Clustered 8-Oxo-Guanine Mutations and Oncogenic Gene Fusions in Microsatellite-Unstable Colorectal Cancer.

PURPOSEColorectal carcinomas (CRCs) with microsatellite-instability (MSI) are enriched for oncogenic kinase fusions (KFs), including NTRK1, RET, and BRAF, but the mechanism underlying this finding is unclear.METHODSThe genomic profiles of 32,218 advanced CRC tumor specimens were analyzed to assess the fusion breakpoints of oncogenic alterations including KFs in microsatellite-stable and microsatellite-unstable CRC. Genomic contexts of such alterations were analyzed to obtain mechanistic insights.RESULTSGenomic analysis demonstrated that oncogenic fusion breakpoints in MSI tumors do not preferentially involve repetitive or low-complexity sequences. Instead, their junction regions showed pronounced guanine and cytosine bias and elevated mutation frequency at G:C contexts. Elevated mutation frequency at G:C bases in relevant introns predicted prevalence of associated oncogenic fusions in MSI CRCs. CRCs harboring mismatch repair signatures had enrichment of butyrate-producing microbial species, reported to be associated with induction of 8-oxoguanine lesions in the intestine.CONCLUSIONDetailed analysis of breakpoints in MSI-associated KFs support a model in which inefficient repair and/or processing of microbiome-induced clustered 8-oxoguanine damage in MSI CRC contributes to the increased incidence of specific oncogenic fusions.

Role of Damage-Associated Molecular Patterns (DAMPs/Alarmins) in Severe Ocular Allergic Diseases.

Severe ocular allergic diseases, such as atopic keratoconjunctivitis and vernal keratoconjunctivitis, cause severe allergic inflammation in the conjunctiva and corneal epithelial damage, resulting in visual disturbances. The involvement of damage (danger)-associated molecular patterns (DAMPs/alarmins) in the pathogenesis of these diseases has been recognized. Alarmins released from damaged corneal epithelial cells or eosinophils play a critical role in the induction of corneal lesions, vicious loop of corneal injury, and exacerbation of conjunctival allergic inflammation. Alarmins in the conjunctiva also play an essential role in the development of both allergic inflammation, based on the acquired immune system, and type 2 inflammation by innate immune responses in the ocular surface. Therefore, alarmins may be a potentially important therapeutic target in severe refractory ocular allergic diseases.

Total flavonoids of sea buckthorn (Hippophae rhamnoides L.) improve MC903-induced atopic dermatitis-like lesions

Ethnopharmacological relevanceSea buckthorn (Hippophae rhamnoides L.) is popularly used as a herbal medicine and food additive in the world. Total flavonoids of Hippophae rhamnoides (TFH) are reported to have anti-inflammatory and immunomodulatory activities. AimThe effects of TFH on atopic dermatitis (AD)-like lesions induced by MC903 in mice was elucidated in the study. MethodsTo induce AD-like lesions, MC903 was adopted to apply repeatedly on the left ear in C57BL/6 mice. After induction of AD-like lesions, 0.5% and 1% TFH cream was applied topically on ears of mice once a day for 8 days. The degree of skin lesions was evaluated by macroscopical and histological methods. Expressions of filaggrin (FLG) was evaluated by Western blotting. Real-time polymerase chain reaction (qPCR) was adopted to detect the mRNA expression of thymic stromal lymphopoietin (TSLP), interferon (IFN)-γ, interleukin (IL-4), tumor necrosis factor (TNF)-α in skin lesions. In vitro, Cytokine Antibody Arrays were performed to measure production of cytokines in IFN-γ/TNF-α-treated HaCaT cells, Western blotting was employed to detect the expressions of p–NF–κB, p-ERK and p-P38. ResultsTopical application of TFH significantly improved the severity of dermatitis by inhibiting the infiltration of mast cell, increasing expression of FLG, decreasing the expressions of TNF-α, IL-4, IFN-γ and TSLP in skin lesions. TFH decreased the levels of IL-1α, IL-1β, IL-6, monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage-derived chemokine (MDC), platelet-derived growth factor (PDGF)-BB, thymus and activation regulated chemokine (TARC) in the supernatants of the HaCaT cells treated by IFN-γ/TNF-α. Furthermore, expressions of p–NF–κB, p-ERK and p-P38 were also decreased by TFH administration with dose dependent manner in HaCaT cells treated by IFN-γ/TNF-α. ConclusionsTopical application of TFH improved AD-like lesions in mice induced by MC903. Which exerted the effects of anti-inflammation and repairing skin barrier by regulating Th1/Th2 balance. This finding indicates that TFH is a novel potential agent for the external treatment of AD.

Evolutionarily conserved bacterial effectors hijack abscisic acid signaling to induce an aqueous environment in the apoplast

High atmospheric humidity levels profoundly impact host-pathogen interactions in plants by enabling the establishment of an aqueous living space that benefits pathogens. The effectors HopM1 and AvrE1 of the bacterial pathogen Pseudomonas syringae have been shown to induce an aqueous apoplast under such conditions. However, the mechanisms by which this happens remain unknown. Here, we show that HopM1 and AvrE1 work redundantly to establish an aqueous living space by inducing a major reprogramming of the Arabidopsis thaliana transcriptome landscape. These effectors induce a strong abscisic acid (ABA) signature, which promotes stomatal closure, resulting in reduced leaf transpiration and water-soaking lesions. Furthermore, these effectors preferentially increase ABA accumulation in guard cells, which control stomatal aperture. Notably, a guard-cell-specific ABA transporter, ABCG40, is necessary for HopM1 induction of water-soaking lesions. This study provides molecular insights into a chain of events of stomatal manipulation that create an ideal microenvironment to facilitate infection.

Cobalt oxide (Co3O4) nanoparticles induced genotoxicity in Chinese hamster lung fibroblast (V79) cells through modulation of reactive oxygen species.

Incessant production, pervasive applications in different fields, and eventually unintended exposure of cobalt oxide nanoparticles (Co3O4 NPs) lead to rise in their toxicity studies toward human health. However, the information regarding the potential toxicity mechanisms of Co3O4 NPs especially genotoxicity is still sparse with missing interconnections. So far, only solitary reports on Co3O4 NPs are at hand, bearing witness to reactive oxygen species (ROS)-mediated DNA damage in lung cells. To address this, we evaluated the Co3O4 NP-induced cytotoxic and genotoxic potential in Chinese hamster lung fibroblast cell line (V79). Our preliminary results demonstrate that Co3O4 NPs at concentrations of 20-100 µg/ml induced moderate mortality after 24-h exposure. However, these low concentrations caused a significant reduction in various organelles' activity in a concentration-dependent manner. Mitochondrial activity and membrane potential were found to be compromised due to NP exposure in a concentration-dependent manner. The study affirms that Co3O4 NPs inhibited lysosomal activity in V79 cells. In addition to this, Co3O4 NPs are also found to stimulate free oxygen radical generation. Genotoxicity studies revealed a potent and dose-dependent effect of non-cytotoxic concentrations of Co3O4 NPs in the induction of DNA lesions. Interestingly, N-acetylcysteine, a free oxygen radical scavenger (5, 10mM, pretreatment) inhibited the progression of free oxygen radicals and induction of Co3O4 NP-mediated DNA lesions. This suggests the ROS-mediated genotoxic potential of Co3O4 NPs.