Mean Optical Density Research Articles

Morpholiogical, quantitative image analysis and molecular biology of oesophageal squamous cell carcinoma (ESCC), with special reference to tumour progression and human papillomavirus (HPV) involvement

It is an urgent need to improve early diagnosis of ESCC, because is one of the most aggressive cancer. The quantitative image analysis can aid in the identification of ESCC. Despite much research effort, the major prognostic factor of ESCC remains the pathological stage of the disease as defined by the TNM classification, whereas tumour grading is of limited value in this respect, mainly due to its low reproducibility. A better means for disease prognostication based on improved understanding of the pathogenetic mechanisms is urgently required. Materials and methods: The material of the present study was derived from a series of 1876 oesophageal surgical specimens taken from a total of 700 patients, who underwent oesophageal resection for an invasive ESCC in Anyang Tumour Hospital, Henan Province of China. Among. The cases of ESCC, previously subjected to extensive testing for Human Papillomavirus (HPV) involvement and expression of p53 gene. All cases are analysed by histopathology and by in situ hybridisation (ISH) and PCR, and a group of 272 patients was randomly selected for analysis of the primary tumour, adjacent mucosa and regional lymph nodes, in the quantitative image analysis. All cases and HPV data were subjected to extensive univariate and multivariate analysis to disclose independent predictors of progressive disease. Results: For the analyses, the ESCCs were graded into two categories: well – moderately and poorly-differentiated. HPV DNA was detected in 116 (18.9 %) of the carcinomas by ISH and in 15.2 % by PCR. In univariate analysis, lymph node status was significantly (p < 0.01) predicted by the following nuclear parameters: nuclear area, G0/G1 ratio, HPV DNA status, integrated optical density (IOD), mean optical density (MOD) and S-Phase. In multivariate analysis, 6 variables remained as independent predictors of disease progression (p < 0.05 level), the three most significant ones being nuclear perimeter, nuclear roundness and equivalent diameter (p < 0.01). Conclusions: A series of quantitatively measured nuclear parameters seem to be a close correlation with ESCC differentiation and progression in univariate analysis and some of these variables proved to be significant independent predictors of disease progression in multivariate modelling as well. These data clearly advocate the use of quantitative image analysis in searching for additional prognostic factors of ESCC.

Comparative Evaluation of Bactericidal Effect of Silver Nanoparticle in Combination with Nd-YAG Laser against Enterococcus faecalis: An In Vitro Study

To evaluate the bactericidal effect of silver nanoparticles and silver nanoparticles in combination with Nd-YAG laser against Enterococcus faecalis. A solution containing 100 μg of silver nanoparticles in 1 mL was prepared by adding 5 mL of 10-4 M solution of AgNO3 with 5 mL of 0.1 M sodium tricitrate. Synthesized silver nanoparticles were characterized using UV-visible spectrophotometer for optical studies and the transmission electron microscopic analysis for determining the size and shape of the nanoparticles. Groups are as follows: group I-silver nanoparticles against E. faecalis, group II-silver nanoparticles in combination with Nd-YAG laser against E. faecalis, group III-control, E. faecalis bacterial culture alone. Optical density was measured periodically at half an hour interval in spectrophotometer in a 96 well plate and statistically analyzed using one-way ANOVA. The optical density and turbidity of groups I and II began to decrease in 2 hours in comparison with the control. There was a significant difference in mean optical density among the three groups after 1½ hours onward. The study also demonstrated the minimal bactericidal concentration (MBC) as 100 μg/mL of Ag nanoparticles with a size of 15 nm were effective against E. faecalis. The study concluded that silver nanoparticles individually and in conjunction with Nd:YAG laser irradiation would be an effective protocol against E. faecalis. The combined effect of silver nanoparticles and laser disinfection against E. faecalis holds a promising treatment modality for eradicating resistant pathogens and biofilms embedded deep inside the dentinal tubules that are not amenable to conventional disinfection protocols in root canals.

Effects of leukocyte-platelet-rich fibrin and advanced platelet-rich fibrin on the viability and migration of human gingival fibroblasts.

Background:Platelet products play a fundamental role in the process of healing. The new generation of platelet-rich fibrin (PRF), namely advanced PRF (A-PRF), has different biological and mechanical properties compared to those of leukocyte-PRF (L-PRF). This study aimed to compare the effects of L-PRF and A-PRF on the viability and migration of human gingival fibroblasts (HGFs).Materials and Methods:In this in vitro study, the effects of A-PRF and L-PRF on the viability and migration of HGFs after 24 and 48 h were evaluated using the methyl thiazolyl tetrazolium assay. The viability of the negative control culture medium was considered to be 100%. The mean optical density of the test groups was divided by that of the negative control group and reported as percentage. One-way ANOVA was applied to assess the effects of time and type of PRF on the viability and migration of HGFs. Pairwise comparisons were made using the Tukey's test.Results:At 24 h, cell viability in the L-PRF group was significantly higher than that in the A-PRF group (P < 0.05). However, no significant difference was noted between the two groups at 48 h. At 24 h, L-PRF caused significantly higher cell migration compared to the negative control group, whereas at 48 h, both A-PRF and L-PRF significantly increased cell migration compared to the control group.Conclusion:Within the limitations of this study, L-PRF and A-PRF had significant effects on the viability and migration of HGFs. Further studies on these platelet concentrates are warranted.

The effect of fine particles on the phagocytosis of alveolar macrophages potentially by Arp2/3 complex in a mouse model of chronic obstructive pulmonary disease

Objective: To investigate the mechanism of fine particulate matter (PM2.5) on the phagocytosis of alveolar macrophages (AM) in mice with chronic obstructive pulmonary disease (COPD) through actin-related protein (Arp) 2/3 complex. Methods: Forty mice were divided into healthy control(A) group, healthy PM2.5 (B) group, COPD(C) group, and COPD PM2.5(D) group according to the random number table method. A mouse model of COPD was established by cigarette smoke exposure method. PM2.5 (662 μg/m(3)) model was established by continuously inhalation for 90 days in healthy PM2.5 group and COPD PM2.5 group. Flow cytometry was used to detect the ability of AM to phagocytose fluorescein isothiocyanate-labeled E.coli (FITC-E.coli), expressed as mean fluorescence intensity (MFI) and percentage of phagocytic positive cells (phagocytosis percentage); Western blotting was used to detect AM Arp2 and F-actin content, and laser confocal microscopy for AM Arp2 and F-actin and phagocytic FITC-E.coli average optical density and colocalization of Arp2 and F-actin, while scanning electron microscopy was used to observes the morphology of AM after phagocytizing FITC-E.coli. Results: AM phagocytosis: MFI and phagocytosis percentage in the COPD group [4 656±251, (31.9±1.7)%] were lower than the healthy control group [8 657±247, (65.7±1.9)%] (both P<0.01); and healthy PM2.5 group and COPD PM2.5 group [7 653±228, (47.9±1.6)% and 3 660±237, (19.2±1.2)%] were lower than the respective control groups (all P<0.01), and the decrease in the COPD group was more pronounced. AM Arp2, F-actin content: the COPD group (0.51±0.02, 0.46±0.03) were lower than the healthy control group (0.81±0.04, 0.71±0.04, both P<0.01); the healthy PM2.5 group and the COPD PM2.5 group [(0.64±0.03, 0.56±0.04) and (0.29±0.02, 0.26±0.02)] were lower than the respective control groups (all P<0.01), and the decrease in COPD group was more significant. Arp2, F-actin, and phagocytic FITC-E.coli mean optical density values: the COPD group (33.0±2.3, 62.0±0.7, 41.0±0.4) were lower than the healthy control group (141.0±4.2, 145.0±2.9, 189.0±2.6, both P<0.01); the healthy PM2.5 group and the COPD PM2.5 group (127.0±2.8, 124.0±0.7, 154.0±0.9, and 24.0±2.4, 37.0±0.4, 29.0±0.8) were lower than the respective control groups (all P<0.01), and the decrease in the COPD group was more significant. Colocalization of AM Arp2 and F-actin: Montessori colocalization coefficient (MOC) (0.38±0.03) in the COPD group was lower than the healthy control group (0.88±0.03, P<0.01); healthy PM2.5 group and COPD PM2.5 group [(0.58±0.03) and (0.14±0.02)] were lower than the respective control groups (both P<0.01), and the decrease in COPD group was more significant. Morphology of AM phagocytosis of FITC-E.coli: AM in the healthy control group was obviously deformed, and the surface of the cell membrane was slightly wrinkled and high, and the free edge of the micro-pleated fold had a long and dense filamentous pseudopodia extension. The changes of morphology of AM in the COPD group was not obvious, the micro-wrinkles on the surface of the cell membrane were rare, and the filopodia poorly extended or even absent. The AM form of the healthy PM2.5 group changed slightly, mostly irregular circular or elliptical. The micro-wrinkles on the surface of the cell membrane were less and flat, and the filopodia protrudes short and less; the AM form of the COPD PM2.5 group was stiff, and the micro-wrinkles on the surface of the cell membrane were few and flat, no obvious filopodia or protrusions. Correlation analysis: After basal state and PM2.5 intervention, AM Arp2, F-actin content and MOC values of Arp2 and F-actin were positively correlated with MFI. Conclusions: The phagocytic function of AM in COPD mice was low, which was related to the abnormal rearrangement of cytoskeleton involved in Arp2/3 complex and F-actin. It was speculated that PM2.5 might inhibit Arp2/3 complex and F-actin. The cytoskeletal rearrangement of proteins was involved in the aggravation of AM phagocytosis in mice with COPD.

The Effects of Adjuvant Tamoxifen Use on Macula Pigment Epithelium Optical Density, Visual Acuity and Retinal Thickness in Patients with Breast Cancer

ABSTRACTPurpose: We aimed to compare best corrected visual acuity, macular pigment optical density and macular thickness in patients with breast cancer, who received oral adjuvant hormone therapy.Materials and Methods: We enrolled consecutive eligible patients with breast cancer who were receiving regular medical tamoxifen treatment. The participants were divided into two groups as cases and controls. Best-corrected visual acuity and retinal thickness were examined. Macular pigment optical density was measured by fundus reflectometry using the one-wavelength reflection method. The output parameters included max optical density, mean optical density, volume and area of the right eye.Results: A total of 104 eyes, cases (n: 50) and controls (n: 54) were included in the study. Mean age in cases was 49.95 ± 9.2 years and 50.21 ± 9.3 years in controls (p = .151). The mean foveal optical density and the maximum optical density differed between cases (0.13 ± 0.03 density units (DU)/0.35 ± 0.07 DU) and controls (0.18 ± 0.04 DU/0.41 ± 0.06 DU) (p = .002/p = .009). Macular pigment optical density volume was 8102.84 ± 2412.67 in cases versus 8280.18 ± 2904.56 in controls (p = .034), and mean MPOD area was 59567.79 ± 11538.06 in cases versus 61748.14 ± 10591.19 in controls (p = .023). The best corrected visual acuity and retinal thickness were similar in both groups (p > .05).Conclusions: Patients in care of oral tamoxifen therapy were found to have significantly reduced macular pigment optical density. In addition, higher drug use duration correlated significantly with reduced macular pigment optical density, suggesting that the poor long-term effects may play a role in macular pigment absorption and incorporation in the retinal tissue.

Evaluation of the role of melatonin in the metastasis of papillary thyroid carcinoma

Objective:To evaluate the level of melatonin and the role of melatonin in the metastasis of papillary thyroid carcinoma in patients with papillary thyroid carcinoma. Method:We measured serum melatonin levels in 81 patients with papillary thyroid carcinoma（PTC） ,20 patients with multinodular goiter（MNG） and 20 healthy adults using ELISA. The relationship between melatonin and clinicopathological features of PTC were analyzed.The expression of MT1 and MT2 in two subtypes of melatonin receptor in 81 cases of papillary thyroid carcinoma and adjacent tissues were detected by immunohistochemical SP method, and its the mean optical density（MOD） image was analyzed by Image Pro Plusversion（IPP） image processing software. Result:Serum melatonin concentration in patients with PTC was significantly higher than that in MNG patients and normal controls（P<0.05）. The level of melatonin in the primary tumor T≥2 cm group was significantly higher than that in the T<2 cm group. Patients with positive cervical lymph nodes（N≥1） had significantly higher melatonin levels than lymph node negatives（N=0）（P<0.05）. The MT1 and MT2 receptors were expressed in both PTC and paracancerous tissues, mainly in the cell membrane and cytoplasm. The expression of MT1 receptor was low in the two groups, and there was no statistical difference. The expression of MT2 receptor in PTC tissues Significantly higher than the adjacent tissues（P<0.05）, further studies showed that the expression of MT2 receptor in PTC tissues was associated with cervical lymph node metastasis, and the expression of MT2 receptor in PTC tissues with cervical lymph node metastasis was significantly lower than that without metastasis （P<0.05）. Conclusion:Serum melatonin levels in PTC patients were higher than those in MNG and control groups, which may be associated with low malignancy of PTC; melatonin inhibits PTC metastasis, which exerts anti-PTC metastasis mainly through MT2 receptors.

INCREASED TRAFFICKING OF MESENTERIC LYMPH-DERIVED γδ T CELLS INTO INTESTINAL MUCOSA IS ASSOCIATED WITH GUT INJURY AFTER INTESTINAL ISCHEMIA-REPERFUSION IN RATS

We sought to investigate the effects of mesenteric lymph-derived γδ T cells trafficking into intestinal mucosa on gut injury after intestinal ischemia-reperfusion (IIR). γδ T cells were separated from mesenteric lymph and then infused into the femoral vein of rats after the γδ T cells were labeled with 51Cr. Migration of γδ T cells in vivo across the intestinal mucosa was determined by γ-counter. Meanwhile, TNF-α activity and endotoxin concentration in mesenteric lymph were detected. The population of γδ T cells of Peyer's patches in the small intestines was analyzed by immunofluorescence double staining methods and flow cytometry. After IIR injury, the mean optical density value (MOD) and population of γδ T cells in Peyer's patches of the gut and migration of 51Cr-γδ T cells across the intestinal mucosa were significantly increased, which had highly positive correlations to degree of intestinal injury, TNF-α levels and endotoxin concentration in mesenteric lymph after reperfusion. The increased population of γδ T cells derived from mesenteric lymph trafficking into the intestinal mucosa might promote the small intestinal injury after IIR.

1369. Clinical Manifestations, Treatment, and Outcome of Nontuberculous Mycobacteria (NTM) Infection in Adult-Onset Immunodeficiency Associated with Anti-interferon-gamma Autoantibodies in King Chulalongkorn Memorial Hospital

BackgroundThere is an increase of anti-interferon-γ autoantibodies syndromes because it is now well recognized, especially in the Southeast Asia region. One of the clinical features is an infection caused by nontuberculous mycobacteria (NTM). NTM infection in patients with anti-interferon-γ autoantibodies is usually severe, recurrent, and disseminated. In this study, we describe the clinical characteristics, treatment, and outcome of NTM infection in patients with anti-interferon-γ autoantibodiesMethodsA retrospective cohort study was conducted at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand, during 2010–2018. Demographic, clinical, and microbiological data were collected and analyzed.ResultsA total of 45 patients were enrolled in this study. Twenty-nine patients were males, and 16 patients were females. The most common clinical manifestations were fever with lymphadenopathy. The median age of the study population was 55 years. The most common organ that was infected was the lymph node (91%). Sweet’s syndrome (68%) was the most common immunological skin reaction. The proportion of infection caused by rapid-growing NTM was more prevalent than slow-growing NTM. The most common NTM was Mycobacterium abscessus (44.4%). The most common co-pathogen identified in this study was Salmonella (37.8%). All patients had positive antibody to interferon-γin the serum which was detected by enzyme-linked immunosorbent assay (ELISA) and mean optical density was 3.28. Fifty-one percent of the patients received long-term antimycobacterial therapy due to chronic active infection, new infections and relapsed. 22.2% of the patients developed a new infection during treatment. However, 28.9% of patients were completely cured of the infection during the follow-up period. The overall mortality rate was 11.1%ConclusionAdult-onset immunodeficiency with anti-interferon-γ autoantibodies is a unique clinical syndrome that has been recognized in Thailand. Treatment of this disorder requires a long duration of the combination of antimycobacterial agents. Sweet’s syndrome is an indicator of recurrence/relapse of NTM infection during the clinical course. Long-term monitoring and immunomodulator are essential for the management of this condition.Disclosures All authors: No reported disclosures.

Hepatitis B infection and risk factors among children living with HIV in Yaounde, Cameroon: an integrated management

BackgroundThe endemicity of hepatitis B virus (HBV) prompted the systematic immunization of newborns in Cameroon since 2005. In the frame of a considerable burden of HIV/HBV co-infection (17.5%), monitoring HBV among children living with HIV (CLHIV) would guide toward HIV/HBV integrated paediatric care. We sought to ascertain the prevalence and determinants of HBV infection in the population of CLHIV and performance of commonly used rapid diagnosis tests (RDTs).MethodsCross-sectional study conducted from February through June 2017 in a subset of CLHIV ≤15 years old at the Essos Hospital Centre, Yaounde, Cameroon. HBV was tested by HBsAg ELISA sandwich in duplicates for each sample, and the mean optical density was calculated. The Determinants of HBV-prevalencewere evaluated, and p < 0.05 was the significance threshold. The performance of two HBV RDTs (Diaspot vs. HBV-5) was evaluated in comparison to ELISA (used as gold standard).ResultsOf the 83 CLHIV enrolled (54.2% female, mean age 8.7 [±3.8] years, 60% vaccinated against HBV, all breastfed), HBV-prevalence was 2.41% (2/83). HBV-positivity was significantly associated with unknown maternal HBV status (2.9% [2/69] vs. 0.0% [0/14], p = 0.0097) and vaginal delivery (2.4% [2/82] vs. 0.0% [0/1], p = 0.0018). Moreover, the most likely to be positive were aged 11 and 15 years, and had experienced neither anti-HBV vaccination nor anti-HBV serum administration, and both had not been treated with any antiseptic solution at birth. Regarding the performance of Diaspot vs. HBV-5 respectively, sensitivity was 100% (2/2) vs. 50% (1/2), while specificity was 100% (45/45) vs. 97.8% (44/45); positive and negative predictive values of Diaspot versus HBV-5 were respectively 100% (2/2) and 100% (45/45) versus 50% (1/2) and 97.8% (44/45).ConclusionHBV-infection in the population of CLHIV appears at a moderate prevalence, suggesting a decreased burden likely due to preventive measures including the wide vaccine coverage. Focusing on mothers with unknown HBV status and promoting safer delivery mode (caesarean section) for HBV-positive motherswould contribute toward pediatric HBV elimination. In context of limited resources, Diaspot test appears more reliable to rollout HBV-infection in the population of CLHIV. As findings are limited to a small sample size, studies on a wider population would be relevant.

Immediate Prone Positioning After Massive Gastric Aspiration Reduces Lung Injury Possibly by Attenuating Interleukin-6-Mediated Lung-Tissue Inflammation in Pigs.

Gastric aspiration, which can cause acute, diffuse, inflammatory lung injury, is of particular concern in critically ill patients. This study aimed to determine the effects of immediate prone positioning on the degree of lung injury and inflammatory response induced by gastric aspiration. Following induction of gastric aspiration by injection of gastric fluid, 16 healthy pigs were randomized to one of two groups: supine position (SP) or prone position (PP). After ventilation and monitoring for 6 hr, all pigs were euthanized. The ratio of the partial pressure of arterial oxygen/fraction of inspired oxygen (PaO2/FIO2) and the partial pressure of arterial carbon dioxide (PaCO2) were recorded during the 6-hr study period. Serum levels of interleukin (IL)-6 were measured every 2 hr, and the mean optical density (MOD) of IL-6 in lung tissues and lung-injury scores were measured at the end of the experiment. The PP group showed a significantly higher PaO2/FIO2 ratio, lower serum IL-6 concentration (p = .015), lower lung-injury scores (p = .012), and lower IL-6 concentration and MOD of IL-6 in lung tissue, especially in dorsal (p = .001, p = .021, respectively) and nondependent regions (p = .005, p = .035, respectively) than the SP group. There were no statistically significant differences in PaCO2 between the groups. Lung-injury severity was positively correlated with the IL-6 concentration and MOD of IL-6 in lung tissues (p < .05). These results suggest that immediate prone positioning alleviated the degree of aspiration-induced lung injury, possibly through mitigating IL-6-mediated lung inflammation.

Comparison of Sensitivity and Specificity of Native ELISA Test (Prepared in Khouzestan, Iran) and Commercial ELISA Kit in the Diagnosis of Human Hydatidosis

Background: As an ancient zoonosis, cystic echinococcosis still is prevalent among livestock worldwide. Early diagnosis is of utmost clinical importance. Objectives: Herein, we compared the efficacy of ELISA with native antigen B and a commercial ELISA kit to detect human hydatidosis in Khuzestan province, southwestern Iran. Methods: The current study consisted of 90 serum samples, including 50 samples obtained from hydatid-affected patients approved by surgery, 20 samples from patients affected by other diseases (having anti-Toxoplasma antibody, giardiasis, hepatitis, etc.), and 20 serum samples from healthy individuals. Native antigen B was prepared from sheep-isolated hydatid cysts. Checkerboard procedure was performed to determine the optimum dilution of antigen, serum and conjugate. Commercial ELISA was done using Vircell indirect immunoenzyme assay to detect anti-hydatidosis IgG. The cut-off point for native ELISA was the sum of two-fold standard deviation and the mean optical density of all negative samples. Results: Using commercial ELISA kit and native ELISA test, 22 out of 90 and 52 out of 90 sera were positive for hydatid-specific IgG, respectively. Compared to the operation, the sensitivity of native and commercial ELISA tests was 100% and 44%, respectively. However, the specificity was not determined due to the lack of surgical information among the heterologous and control groups. In comparison to the commercial ELISA, both tests showed the same sensitivity (97%), while the specificity of native and commercial ELISA was 95% and 96%, respectively. Conclusions: Developing ELISA tests using native antigens would be a reliable method to improve the efficacy of human hydatidosis detection.

Tolvaptan attenuates atrial remodeling in rats undergoing chronic intermittent hypoxia via miRNA-21

Objective: To investigate the effects and potential mechanisms of tolvaptan on chronic intermittent hypoxia (CIH)-induced atrial remodeling in rats. Methods: A total of 45 Sprague-Dawley rats were divided into 3 groups by the random number table: control group, CIH group (6 h/d for 30 days), CIH plus tolvaptan group (8 mg·kg(-1)·d(-1) per gavage for 30 days). Echocardiography examination was performed after 30 days. Thereafter, 5 rats were randomly chosen for histology evaluation, 5 for molecular biological examinations and another 5 rats underwent isolated heart electrophysiology study in each group. Protein and mRNA expression levels of miRNA-21, Spry1, PTEN, ERK/p-ERK, MMP-9, PI3K, AKT/p-AKT were detected. Results: Compared to the rats in control group, rats in the CIH group showed higher atrial interstitial collagen deposition (P<0.001), increased atrial fibrillation inducibility (P=0.022). The results of immunohistochemistry staining showed that the mean optical density (MOD) of ERK, p-ERK and MMP-9 were significantly increased (all P<0.05), the MOD of Spry1 and PTEN were significantly decreased (both P<0.05), above changes could be significantly reversed by cotreatment with tolvaptan. No significant differences were detected in PI3K and AKT among the three groups (P>0.05). In addition, compared with rats in control group, mRNA levels of miRNA-21, MMP-9, PI3K, AKT, and protein levels of ERK, p-ERK, MMP-9 were significantly increased in CIH group(all P<0.05), whereas protein levels of Spry1, PI3K, p-AKT were significantly decreased (all P<0.05). Above changes could be significantly attenuated. Conclusions: CIH induces significant atrial remodeling in this rat model, which can be attenuated by tolvaptan possibly through modulating miRNA-21/Spry1/ERK/MMP-9 and miRNA-21/PTEN/PI3K/AKT signaling pathways.

Efficacy of Recombinant S1 Protein Expressed in Pichia pastoris in Chicken &amp; Using for Detection Titer Antibodies against Avian Infectious Bronchitis by ELISA

Infectious bronchitis (IB) is an acute, extremely contagious upper respiratory disease in chickens. The objective of this study was expressed and using of recombinant S1 Protein serotype 793/B expressed in pichia pastoris in order to serodiagnosis against avian infectious bronchitis antibody. The complete S1 gene (1623bp) was cloned in PTZ57 plasmid and transferred to E. coli (Escherichia coli)-XL1blue bacterium. Next cloned to pPICZB vectors and transferred to p. pastoris(Km71H). Produced protein were visualized by SDS (sodium dodecyl sulfate) PAGE gel that was about with 62KDa. Finally, the S1 recombinant protein was used to coat 96 well plate to recognize antibodies in sera against live Infectious bronchitis virus (IBV 4/91). As a result, this recombinant S1 protein Antigen can be used in an enzyme-linked immunosorbent assay (ELISA) for recognizing antibody titer against IBV. This recombinant protein was evaluated by Elisa, using a panel of field sera for known IBV titer That in the commercial kits, the optical gain of positive control is 0/741 ± 0/007 and the negative control is 0/167 ± 0/002, next the average Optical Density (OD) in the three farms were 2.585, 2.001, 1.657, resulting to the S/P ratio of was 4.212, 2.886, 2.484, respectively. The results of our effort for positive control and the negative control are 0/9 ± 0/010, 0/067 ± 0/008 respectively. The mean Optical Density of sera in 3 Flocks were showed 3.390, 2.737, 2.921, and the ratio of S/P 4. 01, 3.242 and 3.46. These results showed that our outcomes are neared to Biochek kit. The results also showed that recombinant protein S1 was able to identify different kinds of infectious bronchitis virus serotypes in poultry. It can be used for commercial ELISA kit.

Prevalence and outcome of invasive pulmonary aspergillosis in critically ill patients with liver cirrhosis: an observational study

Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity/mortality in critically ill patients with endstage liver disease. Therefore, aim of this study is to predict the prevalence and outcome of IPA in critically ill patients with underlying liver cirrhosis and evaluation of the necessity Glactomannan (GM) screening in serum and bronchoalveolar lavage (BAL) in this cohort. In total 12 out of 84 patients (14%) had probable IPA. The mean optical density index (ODI) bronchoalveolar lavage (BAL) GM index was 3.6 ± 1.5 (Range: 1.7–5.7). An overall sensitivity of 90% (95% CI 86–96%) and specificity of 85% (95% CI 81–88%) was found for the BAL GM in IPA. Acute Physiology And Chronic Health Evaluation (APACHE II), sequential organ failure assessment (SOFA) as well the model of endstage liver disease (MELD) score were significantly higher in the probable IPA group as compared to the No IPA group (26 versus 21, p < 0.001 and 14 versus 10, p < 0.044). Length of intensive care unit (ICU) stay was significantly longer in probable IPA patients (16 versus 10 days, p < 0.027) and mortality rate was significantly higher in probable IPA patients (100% versus 65%, p < 0.001) as compared to No IPA patients. APACHE II and MELD score were independently associated with higher mortality rate using multivariate logistic regression (p = 0.025 and p = 0.034). In conclusion, IPA has a relevant impact on outcome. Screening for IPA is indicated, easy to perform and a necessity to improve outcome.

Effect of actin-related protein 2-3 complex on phagocytosis defect of alveolar macrophages in a mouse model of chronic obstructive pulmonary disease

Objective: To investigate the role of actin-related protein 2-3 complex (Arp2/3) complex on phagocytosis of alveolar macrophages (AMs) in a mouse model of chronic obstructive pulmonary (COPD). Methods: Forty mice were randomly divided into healthy control group, healthy Arp2/3 complex inhibitor (CK666) group, COPD group and COPD CK666 group with 10 mice in each group. COPD group and COPD CK666 group were established by cigarette smoke exposure, and the control group had no smoke exposure. After 90 days of molding, AMs were isolated from lung tissue of mice in each group. Mean fluorescence intensity (MFI) and the positive percent of AMs engulfing fluorescein isothiocyanate-labeled Escherchina coli (FITC-E.coli) (AM%) were detected by flow cytometry. Western blot was applied to detect protein. Laser scanning confocal microscopy was used to measure the mean optical density of Arp2, F-actin and engulfed FITC-E. coli and quantify the colocalization of Arp2 and F-actin by a Manders' overlap coefficient. Scanning electron microscopy was used to observe the ultrastructure of AM phagocytizing FITC-E.coli. Results: Phagocytosis of AM: MFI and AM% in the COPD group were significantly decreased than those in the healthy control group[(4 702±243), (8 684±234) and (32.21±1.66)%, (65.88±1.77)%, all P<0.01]. MFI and AM% in the COPD CK666 group [(3 597±307), (22.09±1.89)%] and in the healthy CK666 group [(7 446±236), (50.09±1.64)%] were decreased compared to those in their respective control groups (all P<0.01). The expressions of protein of Arp2 and F-actin in the COPD group were significantly decreased than those in the healthy control group (0.508±0.025, 0.813±0.040 and 0.462±0.029, 0.720±0.039) (all P<0.01). The F-actin in the COPD CK666 group (0.265±0.014) and in the healthy CK666 group (0.637±0.032) were significantly decreased compared to those in their respective control groups (all P<0.01). The mean optical density of Arp2, F-actin and FITC-E.coli in the COPD group were significantly decreased compared to those in the healthy group (34.43±0.56, 142.83±1.90 and 61.59±0.70, 145.93±3.05 and 41.49±0.33, 189.17±2.60) (all P<0.01); the mean optical density of F-actin, FITC-E. coli in the COPD CK666 group (37.73±1.04, 28.84±2.95) and in the healthy CK666 group (137.07±1.35, 157.46±1.00) were significantly decreased compared to those in their respective control groups (all P<0.01). The Manders' overlap coefficient of Arp2 and phalloidin' coefficient in the COPD group (0.395±0.014) were significantly decreased than the healthy control group (0.395±0.014 and 0.880±0.002, P<0.01). The Manders' overlap coefficient of Arp2 and phalloidin' coefficient in the COPD CK666 group (0.297±0.006) and in the healthy CK666 group (0.737±0.031) were significantly decreased compared to those in their respective control groups (all P<0.01). Shape of AM: Long filopodia protruding and plentiful dorsal ruffle can be seen in AM from the healthy control group; AM pseudopods extension and dorsal ruffle reduced in the health CK666 group; there were pseudopods and dorsal ruffle defects in the COPD group and the COPD CK666 group. Positive correlations existed between the proteins of Arp2, F-actin with MFI. Positive correlations also existed between the Manders' overlap coefficient of Arp2 and phalloidin' coefficient with MFI. Conclusion: Decreased activity of Arp2/3 complex leads to low phagocytosis of AM in COPD mice, and AM in COPD mice is more sensitive to Arp2/3 complex inhibitor.

Age-associated changes of the intrinsic nervous system in relation with interstitial cells in the pre-weaning goat rumen.

In this study, we investigated the neural changes and their relationships with interstitial cells (ICs) in the rumen of pre-weaning goats by transmission electron microscopy, western blot and immunofluorescence (antibody: general neuronal marker-Protein Gene Product (PGP9.5)/ IC marker-vimentin). The immunofluorescence results showed that PGP9.5-positive reaction was widely distributed in neuronal soma (NS) and nerve fibre (NF). The NSs were observed in the ganglia of the myenteric plexus (MP) but not in the submucosal plexus. The mean optical density (MOD) of the whole of PGP9.5-positive nerves and the protein expression level of PGP.5 in the rumen wall both decreased significantly with age. However an obvious increase MOD of PGP.5-positive NFs within the rumen epithelium were observed. In the MP, the nerves and ICs were interwoven to form two complex networks that gradually tightened with age. Furthermore, NSs and nerve trunks were surrounded by a ring-boundary layer consisting of several ICs that became physically closer with aging. Moreover, ICs were located nearby NFs within the ML, forming connections between ICs, smooth muscle cells and axons. This study describes the pattern of neural distribution and its association with ICs in the developing rumen which shed light on the postpartum development of ruminants.

SEROLOGICAL DIAGNOSIS OF BAYLISASCARIS PROCYONIS IN PRIMATES USING A HUMAN ELISA TEST

The usefulness of a human enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Baylisascaris procyonis larva migrans was assessed in nonhuman primates (NHP). The test was originally developed as an assay performed on human samples at Purdue University. Six participating zoos submitted 258 NHP serum samples, spanning these major phylogenetic groups: 1) great apes (n = 84), 2) lesser apes (n = 17), 3) Old World monkeys (n = 84), 4) New World monkeys (n = 20), and 5) prosimians (n = 53). Sera were tested in duplicate using a microtiter-well ELISA with B. procyonis larval excretory-secretory proteins as antigen, and serum from an experimentally infected baboon (Papio anubis) served as positive control. The ELISA clearly identified seropositive animals in all zoos. With putative cutoffs of optical density (OD) measured at 405 nm (OD405) of <0.150 = negative, 0.150-0.250 = indeterminate, and >0.250 = positive, 149 of 258 (57.8%) were clearly negative (mean OD 0.046), and 78 of 258 (30.2%) were clearly positive (mean OD 0.657, range 0.253-1.773), the rest being indeterminate. Of these, 15 were high positive with OD 1.095-1.773 (mean 1.314). Positive animals were seen from all zoos; 76 (97.4%) were great apes, lesser apes, or Old World monkeys. The four highest ODs were in a siamang (Symphalangus syndactylus), lion-tailed macaque (Macaca silenus), Sumatran orangutan (Pongo abelii), and western lowland gorilla (Gorilla gorilla gorilla), all from different zoos. Prosimians had a mean OD of 0.039 and New World monkeys 0.021, indicating that human reagents either did not work for these groups or few infected animals were represented. These results indicate that the human ELISA for B. procyonis works well for at least higher phylogeny NHP and that serologic evidence of infection is surprisingly common, correlating with what is known for exposure to this parasite in zoos.

The expression of NOD2, NLRP3 and NLRC5 and renal injury in anti-neutrophil cytoplasmic antibody-associated vasculitis

BackgroundNucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular sensors of pathogens and molecules from damaged cells to regulate the inflammatory response in the innate immune system. Emerging evidences suggested a potential role of NLRs in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study aimed to investigate the expression of nucleotide-binding oligomerization domain containing protein 2 (NOD2), NOD-like receptor family pyrin domain containing 3 (NLRP3) and NOD-like receptor family CARD domain containing 5 (NLRC5) in kidneys of AAV patients, and further explored their associations with clinical and pathological parameters.MethodsThirty-four AAV patients in active stage were recruited. Their renal specimens were processed with immunohistochemistry to assess the expression of three NLRs, and with double immunofluorescence to detect NLRs on intrinsic and infiltrating cells. Analysis of gene expression was also adopted in cultured human podocytes. The associations between expression of NLRs and clinicopathological parameters were analyzed.ResultsThe expression of NOD2, NLRP3 and NLRC5 was significantly higher in kidneys from AAV patients than those from normal controls, minimal change disease or class IV lupus nephritis. These NLRs co-localized with podocytes and infiltrating inflammatory cells. The mean optical density of NOD2 in glomeruli was significantly higher in crescentic class than non-crescentic class, and correlated with levels of proteinuria and serum creatinine at renal biopsy. The mean optical density of NLRC5 in glomeruli was significantly higher in crescentic class than non-crescentic class, and correlated with proteinuria level, Birmingham Vasculitis Activity Score and the proportion of crescents in the renal specimen.ConclusionsThe expression of three NLRs was upregulated in kidneys of AAV patients. The expression of NOD2 and NLRC5 was associated with the severity of renal lesions in AAV.

Ultrasmall superparamagnetic nanoparticles targeting E-selectin: synthesis and effects in mice in vitro and in vivo.

Purpose: We developed a contrast agent for targeting E-selectin expression. We detected the agent using magnetic resonance imaging (MRI) in vivo in nude mice that had undergone nasopharyngeal carcinoma (NPC) metastasis.Methods: Sialyl Lewis X (sLeX) was conjugated with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. Hydrodynamic size, polydispersity index, and ζ-potential of USPIO–polyethylene glycol (PEG) nanoparticles and USPIO-PEG-sLeX nanoparticles were measured. Component changes in nanoparticles of USPIO, USPIO-PEG, and USPIO-PEG-sLeX were analyzed by thermogravimetric analysis and Fourier-transform infrared spectroscopy. A model of NPC metastasis to inguinal lymph nodes in nude mice was used to investigate characteristics of the USPIO-PEG-sLeX nanoparticles in vivo. We investigated the ability of the T2* value, change in T2* value (ΔT2* value), and enhancement rate (ER) to assess accumulation of USPIO-PEG-sLeX nanoparticles quantitatively in mice of a metastasis group and control group. Four MRI scans were undertaken for each mouse. The first scan (t0) was done before administration of USPIO-PEG-sLeX nanoparticles (0.1 mL) via the tail vein. The other scans were carried out at 0 (t1), 1 (t2), and 2 hours (t3) postinjection. The mean optical density was used to reflect E-selectin expression.Results: sLeX was labeled onto USPIO successfully. In vivo, there were significant interactions between the groups and time for T2* values after administration of USPIO-PEG-sLeX nanoparticles. Six parameters (T2* at t2, ΔT2* at t1, ΔT2* at t2, ER at t1, ER at t2, and ER at t3) were correlated with the mean optical density.Conclusion: USPIO-PEG-sLeX nanoparticles can be used to assess E-selectin expression quantitatively. Use of such molecular probes could enable detection of early metastasis of NPC, more accurate staging, and treatment monitoring.

Emergence of Attenuated Measles Illness Among IgG-positive/IgM-negative Measles Cases: Victoria, Australia, 2008-2017.

Waning measles immunity among vaccinated individuals may result in an attenuated illness. This study compares the epidemiological, clinical, and laboratory profile of measles cases with waning immunity with other measles cases. Polymerase chain reaction-positive (+) measles cases notified to Victoria's Department of Health and Human Services from 2008 to 2017 with immunoglobulin (Ig) M and IgG tested at diagnosis were classified according to serology at diagnosis: IgG negative (-) (nonimmune), IgM+/IgG+ (indeterminate), or IgM-/IgG+ (waning immunity). Between 2008 and 2017, 297 measles cases were notified, of whom 190 (64%) were included; 151 of 190 (79%) were nonimmune at diagnosis, 26 (14%) were indeterminate, and 13 (7%) had waning immunity. Between 2008-2013 and 2014-2017, the proportion of cases with waning immunity increased from 0 of 87 (0%) to 13 of 103 (13%) (P < .001) and the diagnostic sensitivity of initial IgM fell from 93% to 81% (P = .012), respectively. Seven (54%) waning immunity cases reported receiving measles-containing vaccines; 1 case had 2 documented doses. Compared with nonimmune and indeterminate cases, waning immunity cases were more likely to be male; less likely to report fever, coryza, and cough; and had lower burden of virus (higher cycle threshold values). Waning immunity cases had higher IgG titers than indeterminate cases (mean optical density values, 1.96 vs 0.71; P = .004). Onward transmission from 1 waning immunity case was documented. Waning immunity among measles cases, associated with secondary vaccine failure and modified clinical illness, is emerging in Victoria with transmission potential.