Comparison of clinical efficacy of da Vinci robot-assisted lung cancer surgery with two-, three- and four-hole approaches.

Orifice reduction strategies for da Vinci robotic surgery have been a hot topic of research in recent years. We retrospectively analyzed the perioperative outcomes of robotic-assisted thoracoscopic surgery (RATS) with two, three, and four-hole approaches in radical lung cancer surgery. Our results revealed that the two-hole group has advantages in terms of operative time, postoperative 3-day drainage, postoperative drainage time, postoperative hospital stay and postoperative day 3 visual analogue scale (VAS) pain scores. There were no significant differences between the three groups in terms of intraoperative bleeding, number of lymph nodes dissected, VAS pain scores on postoperative days 1 and 2, and postoperative complications. In addition, the two-hole group was superior to the three-hole and four-hole groups in terms of C-reactive protein (CRP), procalcitonin (PCT) and interleukin 10 (IL-10). In summary, the RATS two-hole approach has advantages in operation time, rapid recovery after operation and some postoperative inflammatory indicators, and is worth promoting in hospitals that are skilled in three-hole and four-hole da Vinci robot surgery and have conditions.

[Application Value of Artificial Intelligence-assisted Three-dimensional Reconstruction in Planning Thoracoscopic Segmentectomy].

BACKGROUND: The three-dimensional (3D) can assist in planning lung segmentectomy. 3D reconstruction software based on artificial intelligence algorithm is gradually applied in clinic. The aim of this study was to evaluate the accuracy and safety of 3D reconstruction assisted planning of thoracoscopic segmentectomy. METHODS: A total of 90 patients admitted to Department of Thoracic Surgery of Lanzhou University Second Hospital were evaluated for thoracoscopic segmentectomy. Before operation, artificial intelligence 3D reconstruction software was used to make 3D lung images and conduct preoperative planning. Surgical videos were saved during the operation and perioperative data were recorded. Video recordings of 38 patients were selected to explore the effectiveness of artificial intelligence 3D reconstruction for surgical planning. The results of artificial intelligence 3D reconstruction and Mimics 21 software reconstruction were compared with the actual results in the operation, and the detection and classification ability of bronchus and blood vessels of the two reconstruction methods were compared. RESULTS: All the 90 patients underwent artificial intelligence 3D reconstruction planning, including 57 patients (63.3%) with single lung segmentectomy and 33 patients (36.7%) with combined sub-segmentectomy. The accuracy of artificial intelligence 3D reconstruction for lesion localization was 100.0%, and the accuracy of computed tomography (CT) was 94.4% (85/90). The detection accuracy of artificial intelligence 3D reconstruction and Mimics 21 software was 92.1% (35/38) and 89.5% (34/38), and the anatomic typing accuracy was 89.5% (34/38) and 84.2% (32/38), and the total accuracy was 76.3% (29/38) and 71.1% (27/38). In the comparative observation of 38 surgical videos and reconstructed images, the consistent rates of target segment planning, surgical approach, artery dissection, vein dissection and bronchial dissection for preoperative planning using artificial intelligence 3D reconstruction were 92.1% (35/38), 92.1% (35/38), 89.5% (34/38), 86.8% (33/38) and 94.7% (36/38). The overall planning operational consistency rate was 68.4% (26/38). CONCLUSIONS: It is accurate and safe to use artificial intelligence 3D reconstruction to assist planning thoracoscopic segmentectomy.

[Exploration of the Perturbation of PKIG in Lung Squamous Cell Carcinoma and the Role in Tumor Microenvironment Based on Bioinformatics Method].

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, and patients have limited survival benefits from traditional treatments such as surgery, radiotherapy and chemotherapy. As a new treatment for lung cancer, immunotherapy has significantly prolonged the overall survival (OS) of patients. However, only some patients can benefit from it. We need to explore immunotherapy biomarkers more deeply to screen for advantages. METHODS: The original data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database, and the immunological and prognostic genes of lung squamous cell carcinoma (LUSC) were screened using R software and TIMER database. The expression of target genes was studied in TCGA and GEO databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and correlation analysis with tumor immune characteristics were performed by R software and TISIDB database. RESULTS: We screened out a gene related to immunity and prognosis, cAMP dependent protein kinase inhibitor γ (PKIG), which is significantly differentially expressed in LUSC and normal tissues, and has important reference value for the diagnosis and prognosis assessment of LUSC. PKIG differential genes are mainly concentrated in the regulation of humoral immune response and other processes. The expression of PKIG was positively correlated with the infiltration level of regulatory T cells (Tregs) (r=0.340, P<0.001). In addition, the expression level of PKIG was positively correlated with the expression of chemokines/chemokine receptors such as chemokine C-C motif ligand 2 (CCL2) (r=0.503, P<0.001), CXC chemokine ligand 12 (CXCL12) (r=0.386, P<0.001) and CXC-chemokine receptor 4 (CXCR4) (r=0.492, P<0.001), and immunoinhibitors such as programmed cell death protein 1 (PDCD1) (r=0.359, P<0.001), cytotoxic T-lymphocyte associated antigen 4 (CTLA4) (r=0.375, P<0.001) and T cell immunoglobulin and ITIM domains (TIGIT) (r=0.305, P<0.001) in LUSC. CONCLUSIONS: The immunological and prognostic gene PKIG in lung squamous cell carcinoma was screened through bioinformatics analysis. PKIG is highly correlated with LUSC prognosis and immune microenvironment, and is expected to be a potential biomolecular marker for LUSC immunotherapy.

Estimation of genetic parameters for important traits using a multi-trait model in late-feathering Qingyuan partridge hens in China.

Qingyuan partridge chicken is one of the most well-known Chinese indigenous yellow broilers. In breeding programmes, five traits are usually selected when the chickens are 105 days old, namely body weight (BW), comb height (CH), shank length (SL), shank girth (SG) and feather maturity (FM). The objective of this study was to estimate the genetic parameters of these five traits, especially direct additive genetic correlations, to lay the foundation for balanced selection of Qingyuan partridge chickens. Approximately 9600 records were used for estimation. Variance components for these five traits were estimated using three multi-trait models incorporating different effects via Gibbs sampling. Based on model 1 in which the random effects included direct additive genetic effects and residuals, the estimated direct heritabilities for BW, CH, SL, SG and FM were 0.29 ± 0.04, 0.53 ± 0.04, 0.47 ± 0.04, 0.43 ± 0.05 and 0.18 ± 0.03, respectively. The direct genetic correlations ranged from -0.08 to 0.46. When additionally considering maternal additive genetic effects (model 2), the estimates of direct heritabilities and absolute values of direct additive genetic correlations were smaller. The heritabilities were 0.14 ± 0.04, 0.40 ± 0.02, 0.34 ± 0.05, 0.27 ± 0.05 and 0.12 ± 0.03 for BW, CH, SL, SG and FM, respectively. The direct additive genetic correlations ranged from -0.33 to 0.36. More specifically, the direct additive genetic correlations between BW and CH, SL, SG and FM were 0.19 ± 0.13, 0.15 ± 0.15, 0.36 ± 0.15 and - 0.33 ± 0.21, respectively. The genetic correlations of FM with SL, SG and CH were - 0.15 ± 0.15, -0.08 ± 0.17 and 0.18 ± 0.15, respectively. The direct genetic correlations between CH and SG and SL were - 0.02 ± 0.11 and - 0.20 ± 0.11, respectively, and that between SL and SG was 0.19 ± 0.11. The total heritabilities and maternal additive genetic correlations ranged from 0.16 to 0.44 and from -0.13 to 0.61, respectively. The third model also included the maternal permanent environmental effect for BW. The estimates of direct heritability, direct additive genetic correlation, total heritability and maternal additive genetic correlation were only slightly different from those based on the second model. Therefore, the maternal additive genetic effect has a large effect on the estimation of genetic parameters, and it is better to consider this effect in the genetic evaluation of these five traits. Relatively high direct and maternal additive genetic correlations for most trait pairs suggested that it is better to jointly evaluate these five traits in breeding programmes.

Neoadjuvant sintilimab plus chemotherapy for locally advanced resectable esophageal squamous cell carcinoma: a prospective, single-arm, phase II clinical trial (CY-NICE).

Background: Adding immune checkpoint inhibitors (ICIs) to the chemotherapy has shown significant clinical benefits in neoadjuvant treatment of locally advanced esophageal squamous cell carcinoma (ESCC). Sintilimab is one such ICI used for treatment. Herein, we designed a trial to evaluate the safety and efficacy of sintilimab combined with paclitaxel and platinum for locally advanced resectable ESCC. Methods: Patients with locally advanced resectable (stage II-III) ESCC were enrolled and received at least two cycles of neoadjuvant therapy with sintilimab (200 mg on day 1) plus platinum-based chemotherapy in each 3-week cycle followed by esophagectomy. The primary endpoint of the trial was the pathological complete response (pCR) rate. The secondary endpoints were the major pathological response (MPR) rate, the objective response rate (ORR), the treatment-related adverse events (TRAEs), the immune-related adverse events (irAEs) and quality of life (QOL). Besides, relapse-free survival (RFS), overall survival (OS) were exploratory endpoints. Forty-three cases were needed to be enrolled in this trial. It was assumed the regimen of the neoadjuvant sintilimab plus chemotherapy would achieve a pCR rate of 30.5%. Results: Between March 2021 and January 2023, a total of 43 patients (41 men and 2 women) were enrolled, including 11 cases (25.6%) of clinical stage II and 32 cases (74.4%) of clinical stage III at baseline. All the 43 patients completed two cycles of neoadjuvant therapy, and 32 patients received McKeown radical resection for esophageal cancer. The pCR rate was 28.1% (9/32), which was below the 30.5% reference cutoff value, and the MPR rate was 37.5% (12/32). According to RECIST 1.1, four patients (4/43, 9.3%) had a complete response (CR), 21 patients (21/43, 48.8%) had a partial response (PR), ORR was 58.1% (25/43). The incidence of ≥ grade 3 TRAEs was 23.3% (10/43) and there were no ≥ grade 4 TRAEs. Conclusions: Sintilimab plus platinum-based chemotherapy as neoadjuvant therapy is safe, feasible and effective in locally advanced resectable ESCC, suggesting a supportive rationale for its further evaluation in randomized clinical trials. Trial Registration: Chinese Clinical Trial Registry identifier: ChiCTR2200056558.

SYNGR2 serves as a prognostic biomarker and correlates with immune infiltrates in esophageal squamous cell carcinoma.

BACKGROUND: Synaptogyrin-2 (SYNGR2) plays an important role in regulating membrane traffic in non-neuronal cells. However, the role of SYNGR2 in esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: All original data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and integrated via R 3.5.3. SYNGR2 expression was explored in the TCGA and GEO databases. The correlations between SYNGR2 and cancer immune characteristics were analyzed via the TIMER and TISIDB databases. RESULTS: In general, SYNGR2 was predominantly overexpressed and had reference values in the diagnosis and prognostic estimation of ESCC. Upregulated SYNGR2 was associated with poorer overall survival, disease-specific survival and T stage in ESCC. Mechanistically, we identified hub genes that included a total of 38 SYNGR2-related genes, which were tightly associated with the protein polyubiquitination pathway in ESCC patients. SYNGR2 expression was negatively related to the infiltrating levels of T helper cells. SYNGR2 methylation was positively correlated with the expression of chemokines (CCL2 and CXCL12), chemokine receptors (CCR1 and CCR2), immunoinhibitors (CXCL12 and TNFRSF4) and immunostimulators (CSF1R and PDCD1LG2) in ESCC. CONCLUSION: SYNGR2 may be used as a biomarker for determining prognosis and immune infiltration in ESCC.

[Clinical Study of Artificial Intelligence-assisted Diagnosis System in Predicting the 
Invasive Subtypes of Early-stage Lung Adenocarcinoma Appearing as Pulmonary Nodules].

BACKGROUND: Lung cancer is the cancer with the highest mortality at home and abroad at present. The detection of lung nodules is a key step to reducing the mortality of lung cancer. Artificial intelligence-assisted diagnosis system presents as the state of the art in the area of nodule detection, differentiation between benign and malignant and diagnosis of invasive subtypes, however, a validation with clinical data is necessary for further application. Therefore, the aim of this study is to evaluate the effectiveness of artificial intelligence-assisted diagnosis system in predicting the invasive subtypes of early­stage lung adenocarcinoma appearing as pulmonary nodules. METHODS: Clinical data of 223 patients with early-stage lung adenocarcinoma appearing as pulmonary nodules admitted to the Lanzhou University Second Hospital from January 1st, 2016 to December 31th, 2021 were retrospectively analyzed, which were divided into invasive adenocarcinoma group (n=170) and non-invasive adenocarcinoma group (n=53), and the non-invasive adenocarcinoma group was subdivided into minimally invasive adenocarcinoma group (n=31) and preinvasive lesions group (n=22). The malignant probability and imaging characteristics of each group were compared to analyze their predictive ability for the invasive subtypes of early-stage lung adenocarcinoma. The concordance between qualitative diagnostic results of artificial intelligence-assisted diagnosis of the invasive subtypes of early-stage lung adenocarcinoma and postoperative pathology was then analyzed. RESULTS: In different invasive subtypes of early-stage lung adenocarcinoma, the mean CT value of pulmonary nodules (P<0.001), diameter (P<0.001), volume (P<0.001), malignant probability (P<0.001), pleural retraction sign (P<0.001), lobulation (P<0.001), spiculation (P<0.001) were significantly different. At the same time, it was also found that with the increased invasiveness of different invasive subtypes of early-stage lung adenocarcinoma, the proportion of dominant signs of each group gradually increased. On the issue of binary classification, the sensitivity, specificity, and area under the curve (AUC) values of the artificial intelligence-assisted diagnosis system for the qualitative diagnosis of invasive subtypes of early-stage lung adenocarcinoma were 81.76%, 92.45% and 0.871 respectively. On the issue of three classification, the accuracy, recall rate, F1 score, and AUC values of the artificial intelligence-assisted diagnosis system for the qualitative diagnosis of invasive subtypes of early-stage lung adenocarcinoma were 83.86%, 85.03%, 76.46% and 0.879 respectively. CONCLUSIONS: Artificial intelligence-assisted diagnosis system could predict the invasive subtypes of early­stage lung adenocarcinoma appearing as pulmonary nodules, and has a certain predictive value. With the optimization of algorithms and the improvement of data, it may provide guidance for individualized treatment of patients.

Slanted bilateral lateral rectus recession for convergence insufficiency-type intermittent exotropia: a retrospective study.

BACKGROUND: The present study sought to investigate the efficiency and safety of slanted bilateral lateral rectus recession for the treatment of convergence insufficiency-type intermittent exotropia. METHODS: This retrospective study included 34 patients who underwent slanted bilateral lateral rectus recession for convergence insufficiency-type intermittent exotropia in Shandong Provincial Hospital affiliated to Shandong First Medical University between September 2013 and October 2015 with a minimum follow-up of 6 months. A successful surgical alignment was defined as + 5 (positive for esotropia) to - 10 (negative for exotropia) prism diopters (PD) of orthotropia in the primary position while viewing distant or near targets and a near-distance deviation difference ≤ 8PD. RESULTS: The mean age of the patients at surgery was 7.09 ± 3.80 years (range, 3 to 18 years). The mean distance deviations were - 26.09 ± 6.5 PD (range, - 15 to - 35 PD) and the mean near deviations, - 37.21 ± 6.3 PD (range, - 25 to - 45 PD) preoperatively. The mean recession amount of upper pole of the lateral rectus was 5.97 mm (range, 4.0 to 7.5 mm) and that of lower pole of the lateral rectus, 7.49 mm (range, 6.0 to 8.5 mm). At a mean follow-up of 15.0 months (range, 6 to 37 months), the surgical success rate was 70.6% (24/34), the under-correction rate was 17.6% (6/34), and the overcorrection rate was 11.8% (4/34). The mean near-distance deviation difference was significantly reduced from 11.12 ± 2.06 PD (range, 10 to 15 PD) preoperatively to 2.47 ± 3.04 PD (range, 0 to 10 PD) postoperatively (P < 0.001). Each millimeter of difference between the upper and lower poles of the lateral rectus recession was associated with an improvement of 5.65 PD in the near-distance deviation difference. At the final follow up, a near-distance deviation difference of ≤8PD was found in 32 (94.1%) patients. None of the patients developed A-V pattern, torsional diplopia, or restricted abduction of the eyes. CONCLUSIONS: Slanted bilateral lateral rectus recession may successfully reduce the distance and near exodeviations and the near-distance deviation difference, thus was proved to be an effective and safe procedure for the treatment of convergence insufficiency-type intermittent exotropia.

Intraocular lens optic capture in pediatric cataract surgery.

Posterior capsule opacification (PCO) remains the most common complication of pediatric cataract surgery despite continuous efforts to reduce its incidence. For this reason, pediatric cataract surgeons have expended considerable effort into preventing and mitigating PCO. The intraocular lens (IOL) optic capture technique has been used for the prevention of PCO after pediatric cataract surgery for more than 20y, but there is still no professional consensus. However, recent research has shown encouraging results. The IOL optic capture technique can be performed without anterior vitrectomy to prevent PCO, even in younger children. The type and characteristics of IOLs used for optic capture technique, the location of IOL and the complications of IOL optic capture in children are here reviewed.

Visual outcomes of dense pediatric cataract surgery in eastern China.

PURPOSE: To evaluate the visual outcomes of dense pediatric cataract surgery in eastern China. METHODS: Medical records of children who underwent surgery for dense unilateral or bilateral pediatric cataract in Shandong Provincial Hospital between January 2007 and December 2012 were collected. Patients who cooperated with optical correction and aggressive patching of the sound eye and who had a minimum postoperative follow-up of more than 2 years were included. Risk factors for poor visual outcomes were analyzed. RESULTS: Of the 105 eligible patients (181 eyes), 76 had bilateral cataract, and 29 unilateral. With a mean follow up of 46.77 mo (range 24.0~96.0 mo), the final best corrected visual acuity (BCVA) of 158 eyes were recorded, and 4.43% (7/158) achieved 0.1 logarithm of the minimum angle of resolution (logMAR) or better; 15.19% (24/158) obtained a BCVA between 0.1 logMAR and 0.3 logMAR; 18.99%, (30/158) between 0.3 logMAR and 0.5 logMAR; 46.84% (74/158), between 0.5 logMAR and 1 logMAR; 14.55%, worse than 1 logMAR. The mean BCVA of the patients who underwent lensectomy before 3 months of age was significantly better than that of patients who underwent lensectomy between 3 and 12 months (p = 0.001). In the same lensectomy age groups, the final BCVA of the children in the bilateral and unilateral groups did not differ significantly (P>0.05). Lensectomy after 3 months of age, postoperative complications, strabismus and nystagmus were shown to be risk factors for poor visual outcomes. CONCLUSIONS: Lensectomy before 3 months of age, IOL implantation, proper managing of postoperative complications, early optical correction and aggressive postoperative patching of the sound eye would increase the final BCVA for patients with dense pediatric cataract.

Far distance control scores for assessing intermittent exotropia.

PURPOSE: To assess the level of control in intermittent exotropia by applying the office-based 6-point control scale to far-distance testing. METHODS: Fifty-two children with intermittent exotropia were enrolled in this prospective, noninterventional case series. Control of exodeviation was assessed at indoor distance (ID) of 3 m and near distance of 33 cm using the office-based 6-point control scale, which was additionally applied to indoor far distance (IFD) fixation of 30 m and outdoor far distance (OFD) fixation of 50 m. RESULTS: In all 52 patients, the level of control at OFD fixation was either worse than (31%) or the same as (70%) the level of control at IFD fixation and the level of control at IFD fixation was worse than (64%) or the same as (35%) the level of control at ID fixation. The level of control at OFD fixation was worse than (69%) or the same as (31%) the level of control at ID fixation for all 52 patients. The differences between mean control scores of OFD and IFD fixation (P = 0.002), IFD and ID fixation (P < 0.001), OFD and ID fixation (P < 0.001) were statistically significant. CONCLUSIONS: The outdoor and indoor far distance control scores can increase the sensitivity of the office-based 6-point control scale. The office control scale for assessing control in patients with intermittent exotropia could have application for the management of intermittent exotropia.

Unilateral medial rectus resection for the treatment of recurrent exotropia in children.

PURPOSE: To investigate the outcomes of unilateral medial rectus resection (UMR-res) for the treatment of small to moderate angles of recurrent exotropia in children followed up for a minimum of 6 months. METHODS: This study is a retrospective, consecutive, interventional case series in which 48 children who underwent UMR-res (range 4.0-7.5 mm) for recurrent exotropia [range 12-25 prism diopters (PD)] between January 2009 and February 2013 were enrolled. Of these 48 children, 32 had recurrent intermittent exotropia, and 16 had recurrent constant exotropia. A successful surgical alignment was defined as +5 to -10 PD of orthophoria in the primary position while viewing distant or near targets. RESULTS: At a mean follow-up of 12 months, the surgical success rate was 75 % (36/48), and the undercorrection rate was 25 % (12/48). No patient exhibited overcorrection. The success rates of the UMR-res in the recurrent intermittent exotropia group and recurrent constant exotropia group were 78 % and 69 %, respectively, and were not significantly different (P = 0.50). In the initial surgical procedure groups, the success rates of patients with bilateral lateral rectus recession, unilateral lateral rectus recession, and unilateral lateral rectus recession combined with medial rectus resection were 81.8 % (18/22), 81.25 % (13/16), and 50 % (5/10), respectively. The surgical success rates did not differ among these three groups (P = 0.122). CONCLUSION: Based on our results, UMR-res would appear to be an effective and safe procedure for the treatment of intermittent or constant recurrent exotropia of ≤25 PD in children.

Comparison of different surgery procedures for convergence insufficiency-type intermittent exotropia in children.

AIMS: To compare prospectively the surgical outcomes of different surgery procedures for convergence insufficiency (CI)-type intermittent exotropia (IXT) in children. METHODS: Forty-five children with CI-type IXT were included in this prospective surgical study with 6 months follow-up. According to the different surgical procedures, all children were randomly divided into three groups: the unilateral medial rectus resection (UMR) group (15 cases), the bilateral medial rectus resections (BMR) group (14 cases) and the improved unilateral recession-resection (R&R) group (16 cases). In the UMR and BMR groups, the medial rectus resection(s) were based on the distance exodeviation. In the R&R group, UMR was based on the near exodeviation while lateral rectus recession was based on the distance exodeviation. A successful surgical alignment was defined as the distant deviation in the primary gaze to be between ≤ 10 prism dioptres (PD) of exophoria/tropia and ≤ 5 PD of esophoria/tropia. The success rate, the preoperative and postoperative deviations at distance and near and near-distance differences among groups were compared. RESULTS: At the last follow-up of 6 months, the success rate in the R&R (87.5%) group was significantly higher than those in the UMR (13.3%) and BMR (42.9%) groups (p=0.000 and 0.008); the mean exodeviations at distance and near in the R&R group were significantly different from those in the UMR and BMR groups (p=0.000 and 0.001); there were no significant differences in the mean near-distance differences between the R&R group and the other two groups (p>0.05). CONCLUSIONS: The improved R&R procedure in which medial rectus resection based on the near deviation with lateral rectus recession based on the distant deviation has a better alignment than the UMR and BMR surgeries for the treatment of children with CI-type IXT. All the UMR, BMR and improved R&R surgery can reduce near-distance differences in children with CI-type IXT.

TLR4: the receptor bridging Acanthamoeba challenge and intracellular inflammatory responses in human corneal cell lines.

Acanthamoeba keratitis (AK) is a painful, vision-threatening infection caused by pathogenic strains of the protozoan, Acanthamoeba. Toll-like receptors (TLRs), which are important components of innate immunity, have an important role in the detection of foreign pathogens and the signaling cascades in host cells. However, no report on the interaction between Acanthamoeba and TLR has been found. In this study we analyzed the role of the TLR and its signaling pathway in human telomerase-immortalized corneal epithelial cells (HUCLs) and stromal fibroblasts (THSFs) challenged by Acanthamoeba. We show that the expressions of TLR4, myeloid differentiation protein 88 (MyD88), nuclear factor (NF)-kappaB, phospho-IkappaB, phospho-extracellular signal-regulated kinases 1/2 (p-Erk1/2) and the inflammatory cytokines interleukin (IL)-8, tumor necrosis factor (TNF)-alpha and interferon (IFN)-beta were significantly increased in Acanthamoeba-treated cells. Pretreatment with anti-TLR antibodies or the specific inhibitors pyrrolidine dithiocarbamate (PDTC) (for the NF-kappaB pathway) and U0126 (for the ERK pathway) was conducted. It was found that anti-TLR4 antibody attenuated the production of cytokines induced by Acanthamoeba infection. PDTC inhibited the production of IL-8 and TNF-alpha whereas U0126 inhibited the synthesis of IFN-beta. Thus, TLR4 is a receptor for Acanthamoeba and exerts an effect through TLR4-MyD88-NF-kappaB and TLR4-ERK1/2 pathways to induce the secretion of cytokines in human corneal cell lines challenged by Acanthamoeba.

Evaluation of three different methods to establish animal models of Acanthamoeba keratitis.

PURPOSE: To produce animal models of Acanthamoeba keratitis and to evaluate the advantages and adaptation range of each of the three methods employed. MATERIALS AND METHODS: Mice and Wistar rats in three groups of 15 rats and 15 mice each were used to establish the models. Right corneas in group A were scratched and challenged with Acanthamoeba. Those in group B were scratched and covered with contact lenses incubated with Acanthamoeba. Those in group C received an intrastromal injection of Acanthamoeba. Five rats and 5 mice in each group were used for histopathological investigations and the other 10 in each group were used for clinical evaluation. The models were evaluated by slit lamp examination, microscopic examination and culture of corneal scrapings, HE staining of corneal sections, and pathological scoring of the infections. RESULTS: Four rats and 6 mice in group A, 7 rats and 8 mice in group B, and 10 rats and 10 mice in group C developed typical Acanthamoeba keratitis. CONCLUSION: Corneal scratching alone has the lowest infection rate, while scratching and then covering with contaminated contact lenses has a moderate rate of infection and most closely mimics what happens in most human infections. Intrastromal injection of Acanthamoeba gives a much higher infection rate and more severe Acanthamoeba keratitis.

Expression profiles and function of Toll-like receptors in human corneal epithelia.

BACKGROUND: Toll-like receptors play an important role in the human immune system. This study was conducted to investigate the expression profiles and function of Toll-like receptor (TLR) 1 - 9 in human corneal epithelium. METHODS: The expression of TLR1 - 9 mRNA in 20 human donor corneal epithelia samples abraded during photorefractive keratotomy (PRK) and cultivated telomerase-immortalized human corneal epithelial cells (THCEs) was examined by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Human peripheral blood mononuclear cells (PBMCs) were used as positive controls. The expression of the TLR2 and TLR4 proteins was detected by Western analysis. ELISA was used to detect IL-8 secretion from THCEs challenged with ligands for TLR3 and TLR4 with and without antibody blockade. RESULTS: The expression of TLR1 - 9 at the mRNA level was detected in the epithelia of 20 patients and in THCE. Significant differences among individuals were observed. One patient was found to lack of the expression of TLR3, 4, 6 and 8, whereas another did not express TLR5. The expression of TLR2 and TLR4 protein was detected in human corneal epithelial cells. As THCE cells express TLR1 - 9, cells were challenged with lipopolysaccharides (LPS) and poly I:C to determine whether TLR4 and TLR3 were functional. The results showed that secretion of IL-8 by cells stimulated with LPS and Poly I:C was 7 to 10 fold greater than secretion by unchallenged cells. Blocking TLR4 with an anti-TLR4 antibody significantly inhibited the LPS-induced IL-8 production by THCE (P < 0.05). CONCLUSION: Human corneal epithelial cells express multiple TLRs and are able to recognize LPS and poly I:C. Different expression profiles among individuals suggest that differences in the susceptibilities and sensitivities to bacterial and viral infection in human populations relate to different patterns of TLR expression.

The role of NF-kappaB activation in lipopolysaccharide induced keratitis in rats.

BACKGROUND: Nuclear factor-kappa B (NF-kappaB) is elevated in regulating transcription of many cytokines and inflammatory mediators. The purpose of this study was to investigate the activation and the significance NF-kappaB in lipopolysaccharide (LPS) induced keratitis. METHODS: LPS induced keratitis model was based on Wistar rats. At 0.5, 1, 3, 6, 12, 24 or 72 hours after LPS treatment, the rat corneas were observed with a slit lamp microscope, then the rats were sacrificed and their corneas were excised for routine histological analysis. The expression of NF-kappaB was detected with immunohistochemical staining. The change of tumour necrosis factors-alpha (TNF-alpha) mRNA expression was identified by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Histological findings demonstrated that LPS treated corneas showed significant changes in corneal structure. Corneal edema, pronounced inflammatory cells infiltration and inordinate collagen fibres were observed. Immunohistochemical results showed that the expression of NF-kappaB and its activation obviously increased after LPS treatment compared with the normal group and control group. Positive cells could be observed at 0.5 hour and peak expression of NF-kappaB was observed between 3 hours and 12 hours after infection, but returned to or approached normal level by 72 hours. RT-PCR showed that the level of TNF-alpha mRNA began to increase 0.5 hour after LPS treatment, peaked at 6 hours and then subsided by 72 hours. NF-kappaB had a positive correlation with the expression of TNF-alpha mRNA (r = 0.964, P < 0.01), both NF-kappaB and TNF-alpha had a strong positive correlation with the degree of inflammatory response in LPS treated corneas (r = 0.929, P < 0.01; r = 0.587, P < 0.05, respectively). CONCLUSIONS: The activation of NF-kappaB was increased in LPS treated corneas and was elevated in LPS induced keratitis by promoting overexpression of TNF-alpha mRNA. NF-kappaB may play an important role in the pathogenesis of LPS-associated keratitis in rats.