Advancements in Small Molecule Fluorescent Probes for Superoxide Anion Detection: A Review.

Superoxide anion (O2•-), a significant reactive oxygen species (ROS) within biological systems, plays a widespread role in cellular function regulation and is closely linked to the onset and progression of numerous diseases. To unveil the pathological implications of O2•- in these diseases, the development of effective monitoring techniques within biological systems is imperative. Small molecule fluorescent probes have garnered considerable attention due to their advantages: simplicity in operation, heightened sensitivity, exceptional selectivity, and direct applicability in monitoring living cells, tissues, and animals. In the past few years, few reports have focused on small molecule fluorescence probes for the detection of O2•-. In this small review, we systematically summarize the design and application of O2•- responsive small molecule fluorescent probes. In addition, we present the limitations of the current detection of O2•- and suggest the construction of new fluorescent imaging probes to indicate O2•- in living cells and in vivo.

Clinical phenotype and genetic function analysis of a rare family with hereditary leiomyomatosis and renal cell carcinoma complicated with Birt-Hogg-Dubé syndrome.

To date, over 200 families with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) and over 600 families with Birt-Hogg-Dubé (BHD) syndrome have been reported, with low incidence. Here, we describe a patient with suspected rare HLRCC complicated by BHD syndrome. The proband (II1) had characteristic cutaneous leiomyoma-like protrusions on the neck and back, a left renal mass and multiple right renal, liver and bilateral lung cysts. Three family members (I1, II2, II3) had a history of renal cancer and several of the aforementioned clinical features. Two family members (II1, II3) diagnosed with fumarate hydratase (FH)-deficient papillary RCC via pathological biopsy carried two heterozygous variants: FH (NM_000143.3) missense mutation c.1189G>A (p.Gly397Arg) and FLCN (NM_144997.5) frameshift mutation c.1579_1580insA (p.Arg527Glnfs*75). No family member carrying a single variant had renal tumours. In HEK293T cells transfected with mutant vectors, mRNA and protein expression after FLCN p.Arg527Glnfs*75 and FH p.Gly397Arg mutations were significantly lower than those in wild-type (WT) cells. Cell immunofluorescence showed altered protein localisation and reduced protein expression after FLCN p.Arg527Glnfs*75 mutation. The FH WT was uniformly distributed in the cytoplasm, whereas FH protein expression was reduced after the p.Gly397Arg mutation and scattered sporadically with altered cell localisation. Patients with two variants may have a significantly increased penetrance of RCC.

Microplastics perturb colonic epithelial homeostasis associated with intestinal overproliferation, exacerbating the severity of colitis.

A great amount of the population died due to living or working in an unhealthy environment, highlighting the critical role of environmental pollutants in inducing diseases. Microplastics are widespread environmental pollutants and have been found in various tissues of human beings, yet the risk of microplastics in the occurrence of disease, especially environmentally-related colitis, is unclear. This study focused on the effects of microplastics exposure on intestinal homeostasis and the initiation of colitis. We noticed that microplastics exposure had a limited impact on mice, as verified by no difference observed in bodyweight change, IL-1ß and IL-6 levels in jejunum and liver. Nevertheless, in the colon, the IL-1ß and IL-6 levels were slightly increased and the goblet cell number was decreased. Interestingly, we observed that crypt number and depth, the levels of intestinal stem cell markers, combined with the expression of proliferating cell nuclear antigen and proto-oncogene c-Myc were all significantly increased with microplastics treatment, indicating the overproliferation of colonic mucosa. The effect of microplastics on proliferation and differentiation of crypt was further demonstrated to be regulated by the overactivation of the Notch signaling pathway in intestinal organoids. Furthermore, microplastics exposure accelerated the development of colitis with severe bodyweight loss, diarrhea and bloody stools, macroscopic and pathological damage, and inflammation levels. Worsened liver pathological damage and inflammation in mice with colitis under microplastics exposure also were found. These results suggested that microplastics disrupted the balance between colonic epithelium self-renewal and differentiation, exacerbating the colitis, and might be an environmental-related disease risk factor.

Homochiral organic molecular cage RCC3-R-modified silica as a new multimodal and multifunctional stationary phase for high-performance liquid chromatography.

In this work, homochiral reduced imine cage was covalently bonded to the surface of the silica to prepare a novel high-performance liquid chromatography stationary phase, which was applied for the multiple separation modes such as normal phase, reversed-phase, ion exchange, and hydrophilic interaction chromatography. The successful preparation of the homochiral reduced imine cage bonded silica stationary phase was confirmed by performing a series of methods including X-ray photoelectron spectroscopy, thermogravimetric analysis, and infrared spectroscopy. From the extracted results of the chiral resolution in normal phase and reversed-phase modes, it was demonstrated that seven chiral compounds were successfully separated, among which the resolution of 1-phenylethanol reached the value of 3.97. Moreover, the multifunctional chromatographic performance of the new molecular cage stationary phase was systematically investigated in the modes of reversed-phase, ion exchange, and hydrophilic interaction chromatography for the separation and analysis of a total of 59 compounds in eight classes. This work demonstrated that the homochiral reduced imine cage not only achieved multiseparation modes and multiseparation functions performance with high stability, but also expanded the application of the organic molecular cage in the field of liquid chromatography.

Design of integrated searching and tracking optical systems based on freeform mirrors.

Target searching and tracking photoelectric systems are widely used in civil and military fields. Achieving both efficient search and fine-observation imaging while minimizing the volume and weight of the system is the most concerning problem. In this paper, a dual-mode integrated optical system is proposed, which shares large size components between large field of view (FOV) search module and high-resolution tracking module by using the Biconic Zernike freeform mirrors. Compared with the traditional searching and tracking system utilizing multi-camera matching, this scheme significantly reduces the volume and weight of the remote sensing payload. At the orbit altitude of 500 km, this proposed system has a swath of 87.5 km with a ground resolution of 10 m in search mode. In tracking mode, it can observe an area of 19 km2 with a ground resolution of 1 m. It can meet the requirements of wide-area search and focus tracking simultaneously, providing a feasible scheme for the lightweight design of space optical cameras.

Preparation and chiral resolution properties of bridged bis(cyclodextrin)s hybrid spheres for high performance liquid chromatography.

Selenium-bridged bis(ß-cyclodextrin)s organic-inorganic hybrid silica material with regular spherical shape as new type of chiral stationary phase was directly synthesized under the one-pot hydrothermal synthesis method, and the chiral stationary phase was further characterized by infrared spectroscopy, scanning electron microscopy, thermogravimetry, and elemental analysis. The results of chiral separation showed that eight chiral compounds including various types of chiral alcohols and flavanone were successfully separated in the reversed-phase separation mode by high performance liquid chromatography, which showed the better chiral resolution effect than that on the C2 position of single ß-cyclodextrin. The mechanism of chiral separation was likely due to multiple interactions such as inclusion, hydrogen bonding, electrostatic interaction, dipole-dipole interaction, and the synergistic effect of two cyclodextrins during the chiral resolution process. The synergy of the two cyclodextrins has great potential for development in chiral resolution.

Clinical and imaging features of six Han patients with SAPHO syndrome.

BACKGROUND: Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a rare autoimmune disease characterized by skin or osteoarticular damage. SAPHO syndrome is often misdiagnosed or missed diagnosis due to lack of overall understanding of the disease by clinicians. PURPOSE: To analyze the clinical symptoms and imaging features of six Han patients with SAPHO syndrome in order to provide reference for doctors to diagnose SAPHO syndrome. MATERIAL AND METHODS: This study retrospectively analyzed the clinical data of six Han patients with SAPHO syndrome. RESULTS: All six Han patients with SAPHO syndrome had severe acne or pustulosis of the hands and feet, and all of them had osteoarticular damage, including five cases involving the sternoclavicular joint. Some patients showed a specific and typical "bull's head" sign on 99mTc-labeled methylene diphosphonate bone imaging. Among the six patients recruited, there was one thoracic vertebra, one cervical vertebra, one sacroiliac joint, and one peripheral joint involvement. Two patients had limited activity due to severe osteoarticular damage. CONCLUSION: Due to the atypical clinical symptoms of SAPHO syndrome, most patients will experience a tortuous and long diagnostic process, while a correct understanding and timely intervention of SAPHO syndrome are essential to improve the prognosis of patients.

Porous Organic Cage-Embedded C10-Modified Silica as HPLC Stationary Phase and Its Multiple Separation Functions.

Reduced imine cage (RCC3) was covalently bonded to the surface of silica spheres, and then the secondary amine group of the molecular cage was embedded in non-polar C10 for modification to prepare a novel RCC3-C10@silica HPLC stationary phase with multiple separation functions. Through infrared spectroscopy, thermogravimetric analysis and nitrogen adsorption-desorption characterization, it was confirmed that RCC3-C10 was successfully bonded to the surface of silica spheres. The resolution of RCC3-C10@silica in reversed-phase separation mode is as high as 2.95, 3.73, 3.27 and 4.09 for p-phenethyl alcohol, 1-phenyl-2-propanol, p-methylphenethyl alcohol and 1-phenyl-1-propanol, indicating that the stationary phase has excellent chiral resolution performance. In reversed-phase and hydrophilic separation modes, RCC3-C10@silica realized the separation and analysis of a total of 70 compounds in 8 classes of Tanaka mixtures, alkylbenzene rings, polyphenyl rings, phenols, anilines, sulfonamides, nucleosides and flavonoids, and the analysis of a variety of chiral and achiral complex mixtures have been completed at the same time. Compared with the traditional C18 commercial column, RCC3-C10@silica exhibits better chromatographic separation selectivity, aromatic selectivity and polar selectivity. The multifunctional separation mechanism exhibited by the stationary phase originates from various synergistic effects such as hydrophobic interaction, π-π interaction, hydrogen bonding and steric interaction provided by RCC3 and C10 groups. This work provides flexible selectivity and application prospects for novel multi-separation functional chromatographic columns.

Regulation of the Paneth cell niche by exogenous L-arginine couples the intestinal stem cell function.

Although previous studies show that exogenous nutrients regulate the stem cell function, little is known about the effects of L-arginine on intestinal stem cells (ISCs). In this study, we utilize mice, small intestinal (SI) organoids, and ISC-Paneth cell co-cultured models to clarify the role of L-arginine in ISC function. We find that exogenous L-arginine is essential for ISCs proliferation and intestinal epithelial renewal. Our data show that Paneth cells, a critical component of the ISCs niche, augment the ISCs function in response to L-arginine. Moreover, enhanced the expression of Wnt3a in Paneth cells, which is a ligand of the Wnt/ß-catenin signaling pathway, mediates the effects of L-arginine on ISCs function. Pre-treatment with L-arginine enhances the ISCs pool and protects the gut in response to injury provoked by murine tumor necrosis factor α (TNF-α) and 5-Fluorouracil (5-FU). Our findings establish that the regulation of Wnt3a in the Paneth cell niche by exogenous L-arginine couples ISCs function and favours a model in which the ISCs niche couples the nutrient levels to ISCs function.

Akkermansia muciniphila protects intestinal mucosa from damage caused by S. pullorum by initiating proliferation of intestinal epithelium.

Akkermansia muciniphila, a novel mucin-degrading bacterium, has been demonstrated to prevent the development of obesity and related complications. However, whether it can protect poultry from intestinal mucosal damage by enteropathogens has never been mentioned. In this study, we found that A. muciniphila colonized in the intestine and then relieved intestinal mucosal damage in chicks caused by S. pullorum, including anatomical and morphological damage, alleviation of body weight and intestinal inflammation. The repair process activated by A. muciniphila is accompanied by an increase in the number of goblet cells in the chick's intestine and an up-regulation of Mucin 2 and trefoil factor 2 (Tff2). In addition, we also demonstrate that A. muciniphila improved colon length, crypt depth, increased the proliferating cell nuclear antigen, with the accelerated proliferation of intestinal epithelium through Wnt/ß-catenin signaling pathway, thereby restoring the damaged intestinal mucosa. This study suggests that A. muciniphila activates the proliferation of intestinal cells protecting the intestinal barrier, thus relieving infection with S. pullorum in chickens.

Three-dimensional printing technology for localised thoracoscopic segmental resection for lung cancer: a quasi-randomised clinical trial.

BACKGROUND: Three-dimensional (3D) computed tomography (CT) reconstruction technology has gained attention owing to its potential in locating ground glass nodules in the lung. The 3D printing technology additionally allows the visualisation of the surrounding anatomical structure and variations. However, the clinical utility of these techniques is unknown. This study aimed to establish a lung tumour and an anatomical lung model using 3D printing and 3D chest CT reconstruction and to evaluate the clinical potential of 3D printing technology in uniportal video-assisted thoracoscopic segmentectomy. METHODS: Eighty-nine patients with ground glass nodules who underwent uniportal video-assisted thoracoscopic segmentectomy were classified into the following groups: group A, lung models for pre-positioning and simulated surgery that were performed with 3D chest CT reconstruction and 3D printing, and group B, patients who underwent chest CT scans with image enhancement for 3D reconstruction. The differences in the surgery approach transfer rate, surgical method conversion rate, operative time, intraoperative blood loss, and postoperative complication rate were compared between the two groups. RESULTS: Between groups A and B, there were significant differences in the approach transfer rate (0% vs.10.5%, p = 0.030), operative time (2.07 ± 0.24 h vs. 2.55 ± 0.41 h, p < 0.001), intraoperative blood loss volume (43.25 ± 13.63 mL vs. 96.68 ± 32.82 mL, p < 0.001) and the rate of surgical method conversion to lobectomy (0% vs. 10.5%, p < 0.030). In contrast, there was an insignificant difference in the postoperative complication rate between groups A and B (3.9% vs. 13.2%, p = 0.132). CONCLUSIONS: 3D printing technology facilitates a more accurate location of nodules by surgeons, as it is based on two-dimensional and 3D image-based findings, and therefore, it can improve surgical accuracy and safety. This technique is worth applying in clinical practice.

Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1ß (IL-1ß), IL-6, IL-8, transforming growth factor ß (TGF-ß), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-ß is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection.IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection.

AKT isoform-specific expression and activation across cancer lineages.

BACKGROUND: Aberrant AKT activation is prevalent across human cancer lineages, providing an important therapeutic target. AKT comprises three isoforms that mediate critical non-redundant, even opposing functions in cancer pathophysiology. Therefore, targeting specific AKT isoforms in particular cancers may be more effective than pan-AKT inhibition while avoiding disadvantages of pan-AKT inhibition. Currently, AKT isoform-specific expression and activation in cancer are not clearly characterized. METHODS: We systematically characterized AKT isoform-specific expression and activation in 211 cancer cell lines derived from different lineages and genetic backgrounds using a reverse-phase protein array platform. RESULTS: We found that phosphorylation, but not expression, of AKT1 and AKT2 was coordinated in most but not all cells. Different cancer lineages displayed differential AKT1 and AKT2 expression and phosphorylation. A PIK3CA hotspot mutation H1047R but not E545K was associated with selective activation of AKT2 but not AKT1. CONCLUSIONS: Our study identified and validated AKT isoform-specific expression and phosphorylation in certain cell lines and demonstrated that genetic changes can affect AKT isoform-specific activation. These results provide a more precise understanding of AKT isoform-specific signaling and, in addition, facilitate AKT isoform targeting for personalized cancer therapies.

System design for a "checkerboard" imager.

In order to solve the problem of uneven spatial frequency sampling and lack of zero frequency sampling in the segmented planar imaging detector for electro-optical reconnaissance (SPIDER), we propose a "checkerboard" imaging system design using a square grid aperture arrangement, show its aperture matching method, and demonstrate that it can achieve the uniform sampling of all integer multiples of fundamental frequency in the highest frequency range with zero frequency in the direction of two orthogonal arrays. Through computer simulation, it is verified that the imaging effect of the "checkerboard" imaging system is superior to that of the SPIDER imaging system and the traditional single aperture imaging system at the same system diameter.

Crosstalk between H9N2 avian influenza virus and crypt-derived intestinal organoids.

The spread of Avian influenza virus via animal feces makes the virus difficult to prevent, which causes great threat to human health. Therefore, it is imperative to understand the survival and invasion mechanism of H9N2 virus in the intestinal mucosa. In this study, we used mouse threedimensional intestinal organoids that contained intestinal crypts and villi differentiated from intestinal stem cells to explore interactions between H9N2 avian influenza virus and the intestinal mucosa. The HA, NA, NP and PB1 genes of H9N2 viruses could be detected in intestinal organoids at 1 h, and reached peak levels at 48 h post-infection. Moreover, the HA and NP proteins of H9N2 virus could also be detected in organoids via immunofluorescence. Virus invasion caused damage to intestinal organoids with reduced mRNA transcript expression of Wnt3, Dll1 and Dll4. The abnormal growth of intestinal organoids may be attributed to the loss of Paneth cells, as indicated by the low mRNA transcript levels of lyz1 and defcr1. This present study demonstrates that H9N2 virus could invade intestinal organoids and then cause damage, as well as affect intestinal stem cell proliferation and differentiation, promoting the loss of Paneth cells.

Necrotizing soft tissue infection: clinical characteristics and outcomes at a reconstructive center in Jilin Province.

BACKGROUND: To investigate the clinical characteristics and treatment outcomes in necrotizing fasciitis (NF) patients in a reconstructive unit in northeastern China. METHODS: Medical records of patients diagnosed with and treated for NF in the extremities from November 2013 to December 2016 were retrospectively reviewed. Demographic data, clinical presentation, duration of signs and symptoms, location of infection, predisposing factors, causative microbiological organisms, laboratory risk indicator for necrotizing fasciitis (LRINEC) score, number of surgical debridements, length of hospital stay, treatments, and outcomes were recorded. RESULTS: A total of 39 consecutive patients were treated for severe NF (32 male and 7 female). Diabetes mellitus and blunt trauma were the most common risk factors (13 and 9 cases, respectively). The positive predictive value of the LRINEC score in NF diagnosis was 46.2%. Mean duration of signs and symptoms was 4.6 days. Staphylococcus aureus was the most commonly isolated bacteria (20 cases). All patients underwent their first debridement within 12 h of presentation (mean, 4.6 h). Mean number of surgical treatments was 2.8 (range, 2-5) per patient, including debridements. All patients survived, and mean length of hospital stay was 30.81 (range, 21-43) days. Three patients underwent limb amputation. CONCLUSIONS: In our clinical experience, early detection and aggressive debridement are the cornerstones of NF treatment. Antibiotic therapy and intensive care support is essential in severe cases of NF. Anaerobic tissue culture and frozen section biopsy could be adopted as routine tests for diagnosis and decision-making in NF. These findings should inform clinical decisions about the treatment of individual patients with NF.

Couple-kissing flaps for successful repair of severe sacral pressure ulcers in frail elderly patients.

BACKGROUND: Surgical repair of severe pressure ulcers (PUs) in elderly patients remains a challenge for clinicians due to the complicated comorbidities and the special physical characteristics of elderly patients. The objective of this study was to evaluate the application of couple-kissing flaps (CKF) in the reconstruction of sacral PUs in these patients. METHODS: Elderly patients (over 70 years) with stage 3 or stage 4 PUs who underwent CKF immediately after radical debridement between July 2012 and December 2015 were enrolled in this retrospective study. Patients' demographics were extracted from the medical records. RESULTS: A total of 12 patients were involved in this study. The average age of the patients was 76.83 years (ranged from 71 to 92 years). The donor site was closed primarily in all cases. All the flaps healed uneventfully without complications. Follow-up observations were conducted for an average of 13.6 months (ranged from 9 months to 2 years). Cosmetic results were satisfactory, with no surgical site breakdown or recurrence of PU in any of the cases. Three representative cases are presented. CONCLUSIONS: The CKF is a reliable and satisfactory option for the reconstruction of severe sacral PUs defects in elderly patients. CKF is associated with an relatively low rate of complications and recurrence.

Comparison of nanofluoridated hydroxyapatite of varying fluoride content for dentin tubule occlusion.

PURPOSE: To evaluate the viability of a series of nano-fluoridated hydroxyapatite (nano-FHA) formulations of varying fluoride content for the occlusion of exposed dentin tubules, in comparison to nano-hydroxyapatite (nano-HA). METHODS: Nano-FHA powders with varying levels of fluoride ion were synthesized to substitute hydroxyl ions (⁻OH) present in hydroxyapatite (HA). Nano-FHA were defined as 2nFHA,4nFHA,6nFHA, 8nFHA and 10nFHA, with the molar ratio of F(⁻ to Ca243; of 0.002,0.004,0.006,0.008 and 0.01, respectively. The powders were synthesized using a wet chemistry route, and characterized by transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The methylthiazol tetrazolium (MTT) assay was used to assess cell viability toward nano-FHA. According to the content of F⁻, the nano-nFHA and nano-HA powders were divided into six groups, with the artificial saliva as control. They were spread over the dentin surface for 1 minute and repeated three times per day for 7 consecutive days. After washing and brushing with distilled water for an additional 7 days, scanning electron microscopy was used to evaluate the in vitro plugging rate of the tubules and penetrating depth. RESULTS: The 2nFHA, 4nFHA, 6nFHA, 8nFHA and 10nFHA powders were prepared and characterized. The 8nFHA had less crystallinity compared to 2nFHA,4nFHA, and 6nFHA.The MTT assay (from 12 to 120 hours) showed that the cell viability of the L-929 fibroblasts in the 2nFHA, 4nFHA, 6nFHA, 8nFHA, 10nFHA, and nano-HA groups ranged from 80.54± 3.35% to 112.9± 4.8%. Most of the nano-FHA powders successfully occluded dentin tubules. The plugging rate of 8nFHA was significantly higher than that of the 2nFHA, 4nFHA, 6nFHA and nano-HA groups. The penetrating depth of the 8nFHA group into the tubules was also significantly higher than that of the other nano-FHA and nano-HA group. CLINICAL SIGNIFICANCE: The nano-FHA formulation 8nFHA showed higher plugging rate and penetrating depth into the tubules. It has the potential to be used as a desensitizing agent in treating dentin hypersensitivity with better long-term durability and efficacy of dentin tubule occlusion.

CpG Oligodeoxynucleotides Facilitate Delivery of Whole Inactivated H9N2 Influenza Virus via Transepithelial Dendrites of Dendritic Cells in Nasal Mucosa.

UNLABELLED: The spread of the low-pathogenicity avian H9N2 influenza virus has seriously increased the risk of a new influenza pandemic. Although whole inactivated virus (WIV) vaccine via intranasal pathway is the effective method of blocking virus transmission, the mucosal barrier seems to be a major factor hampering its development. CpG oligodeoxynucleotides, a known adjuvant, can target downstream dendritic cells (DCs) and effectively enhance the mucosal and systemic immune responses. However, the ability of CpGs to assist H9N2 WIV in transepithelial transport remains unknown. Here, in vitro and in vivo, we showed that CpGs provided assistance for H9N2 WIV in recruiting DCs to the nasal epithelial cells (ECs) and forming transepithelial dendrites (TEDs) to capture luminal viruses. CD103(+) DCs participated in this process. Chemokine CCL20 from nasal ECs played a key role in driving DC recruitment and TED formation. Virus-loaded DCs quickly migrated into the draining cervical lymph nodes (CLNs) for antigen presentation. In addition, the competence of CpGs was independent of direct epithelial transport via the transcellular or paracellular pathway. Taken together, our data demonstrated that CpGs enhanced the transport of H9N2 WIV via TEDs of nasal DCs, which might be a novel mechanism for optimal adaptive immune responses. IMPORTANCE: This paper demonstrates by both an in vivo and an in vitro coculture model that CpG oligodeoxynucleotides, known as an adjuvant generally targeting downstream immune responses, also are crucial for the transport of H9N2 WIV across nasal epithelial cells (ECs) via the uptake of transepithelial dendrites (TEDs). Our results prove for the first time to our knowledge that the immune-potentiating mechanism of CpGs is based on strengthening the transepithelial uptake of H9N2 WIV in nasal mucosa. These findings provide a fresh perspective for further improvement of intranasal influenza vaccines, which are urgently needed in the face of the potential threat of H9N2 influenza.

Effects of arginine on intestinal epithelial cell integrity and nutrient uptake.

Arginine is a multifaceted amino acid that is critical to the normal physiology of the gastrointestinal tract. Oral arginine administration has been shown to improve mucosal recovery following intestinal injury. The present study investigated the influence of extracellular arginine concentrations on epithelial cell barrier regulation and nutrition uptake by porcine small intestinal epithelial cell line (IPEC-J2). The results show that reducing arginine concentration from 0·7 to 0·2 mm did not affect the transepithelial electrical resistance value, tight-junction proteins (claudin-1, occludin, E-cadherin), phosphorylated extracellular signal-regulated protein kinases (p-ERK) and mucin-1 expression. Furthermore, reducing arginine concentration stimulated greater expression of cationic amino acid transporter (CAT1), excitatory amino acid transporter (EAAT3) and alanine/serine/cysteine transporter (ASCT1) mRNA by IPEC-J2 cells, which was verified by elevated efficiency of amino acid uptake. Glucose consumption by IPEC-J2 cells treated with 0·2 mm-arginine remained at the same physiological level to guarantee energy supply and to maintain the cell barrier. This experiment implied that reducing arginine concentration is feasible in IPEC-J2 cells guaranteed by nutrient uptake and cell barrier function.