Reverse Transcription-polymer Chain Reaction Research Articles

Gold nanoparticle dimer-based immunochromatography for in situ ultrasensitive detection of porcine epidemic diarrhea virus.

The low detection sensitivity of lateral-flow immunochromatography assay (LFIA) based on spherical gold nanoparticle (AuNP) limits its wide applications. In the present study, AuNP dimers with strong plasma scattering and robust signal output were synthesized via the Ag ion soldering (AIS) strategy and used as labeled probes in LFIA to boost the sensitivity without any extra operation process and equipment. The established LFIA exhibited high sensitivity with a limit of detection (LOD) of 2.0 × 102 TCID50/mL for PEDV, which provides50 times highersensitivity than commercial LFIA based on spherical colloidal gold. In addition, the AuNP dimer-based LFIA showed strong specificity, good reproducibility, high stability, and good accordance to reverse transcription polymer chain reaction (RT-PCR) when detecting 109 clinical samples. Thus, the AuNP dimers is a promising probe for LFIA and the developed AuNP dimer-based LFIA is suitable for the rapid detection of PEDV in the field.

Cough Audio Analysis for COVID-19 Diagnosis.

Humanity has suffered catastrophically due to the COVID-19 pandemic. One of the most reliable diagnoses of COVID-19 is RT-PCR (reverse-transcription polymer chain reaction) testing. This method, however, has its limitations. It is time consuming and requires scalability. This research work carries out a preliminary prognosis of COVID-19, which is scalable and less time consuming. The research carried out a competitive analysis of four machine-learning models namely, Multilayer Perceptron, Convolutional Neural Networks, Recurrent Neural Networks with Long Short-Term Memory, and VGG-19 with Support Vector Machines. Out of these models, Multilayer Perceptron outperformed with higher specificity of 94.5% and accuracy of 96.8%. The results show that Multilayer Perceptron was able to distinguish between positive and negative COVID-19 coughs by a robust feature embedding technique.

Functional activity of mitochondria associated with cytoskeleton stability in the HEK293

Mitochondria are dynamic cellular organelles that provide the biological energy to the cell. The dynamic property of the mitochondria is associated with the cytoskeleton network in the cell. However, relationship is no clear identified between cytoskeleton instability and the functional activity of mitochondria in cell. This study is designed to assess the dynamic properties of the mitochondria, and specifically to observe whether they are associated with the cytoskeleton stability for the mitochondria’s functional activityWe prepared the cytoskeleton stability regulation model with the HEK293 cell line by vinorelbine which microtubule distributor and the cytoskeleton stabilizer as a taxol. We determined the optimal concentration of vinorelbine and taxol though cytotoxicity assay with the HEK293 cell line. Then, we analyzed the functional activity of the mitochondria depending on the microtubule instability. First, we undertook an observation of cell morphology under distributor or stabilizer. We analyzed the metabolic activity of the cell with the CCK and ATP productive assay. And we performed confocal live imaging for observation of mitochondria morphology in the cytoskeleton instable cells. We performed the reverse transcription polymer chain reaction for analysis of the mRNA expression in the cytoskeleton’s instable cells with Tau protein, Glut1 and GAPDH as the normalization marker.The vinorelbine treated cell shows dramatically changes with cell morphology compared with control. And the vinorelbine treated cell significantly increases the fission ratios of the mitochondria compared with the original form based on the confocal images. Microtubule instable cells significantly decreased the ATP’s productive ratios and the functional activity of the mitochondria. However, the cytoskeleton stabilizer as a taxol(0.01uM) combined with vinorelbine treated cells, shows recovery effect to cell morphology recovery effect like the control cells that were only treated with vinorebine. And taxol that was combined with vinorelbine treated cells significantly increased bioenergy product ratios and its functional activity compared with the vinorelbine.In conclusion, the dynamic mitochondrial motility might influence the microtubule stability in cells. Because, vinorelbine‐treated cells displayed destabilized microtubules, loss of motility and fission formation of mitochondria. And loss motility of the mitochondria shows poor ATP productive ratios, resulting in a phenotype like that observed in the cells. This data suggests that functional activity of the mitochondria is associated with the microtubule stability in the cells. And microtubule stabilizer has a possibility to recovery functional activity in the microtubule instable cell like aging process. This finding can be applying to understand the mechanism of aging.Support or Funding InformationThis research was supported by the University Innovation Support Project (UISP) through the National Research Foundation (NRF) funded by MEST(Project No. 20190104)CCK analysis(A) and cell morphology(B) of NaB and Taxol treated cellsFigure 1ATP productive ratios(A) and fission form of mitochondria(B) of cellsFigure 2

Expression and correlation of Fra-1 and HMGA1 in laryngeal squamous cell carcinoma

Objective: To investigate the expressions of Fra-1 and HMGA1 in laryngeal squamous cell carcinoma and their correlation. Methods: Immunohistochemistry and reverse transcription-polymer chain reaction (RT-PCR) were used to detect the expressions of HMGA1 and Fra-1 in laryngeal squamous carcinoma tissues in 47 cases and para-carcinoma tissues in 21 cases(the First Hospital of Shijiazhuang). The relationship between the gene expressions in carcinoma tissues and clinopathological parameters such as pathological grade, clinical stage, lymph metastasis, age and anatomic site and the relevance of the two gene expressions were analyzed. SPSS 13.0 software was used to analyze the data. Results: The positive expression rates of Fra-1 and HMGA1 proteins in laryngeal squamous cancer tissue were 48.9% and 53.2%, which were respectively higher than the rates of 19.0% for Fra-1 (χ(2)=5.416, P<0.05) and of 23.8% for HMGA1 (χ(2)=5.083, P<0.05) in adjacent tissues. The expression of Fra-1 gene was correlation with pathological grade, clinical stage and lymph metastasis (t values were -1.079, -1.066 and -1.067, all P<0.05), but not with age and anatomic site (t values were -1.068 and -1.054, both P>0.05). The expression of HMGA1 gene was correlation with pathological grade, clinical stage, lymph metastasis and age (t values were -1.112, -1.065, -1.009 and -1.066, all P<0.05), but not with anatomic site (t=-1.036, P>0.05). The expressions of Fra-1 and HMGA1 gene were positively correlation (r=0.672, P<0.05). Conclusions: In laryngeal squamous cancer, Fra-1 and HMGA1 are excessive expression, with a positive correlation between the expressions of both genes.

Quantitative Analysis of Milk-Derived microRNAs and Microbiota during the Manufacturing and Ripening of Soft Cheese.

MicroRNAs (miRNAs) are abundant in bovine milk and milk derived from other livestock, and they have functional roles in infants and in the secretion process of mammary glands. However, few studies have evaluated miRNAs in dairy processes, such as during cheese making and ripening. Thus, we investigated the characteristics of milk-derived miRNAs during the manufacturing and ripening of Camembert cheese as well as the microbiota present using the quantitative reverse transcription polymer chain reaction (RT-qPCR) and 16S rRNA pyrosequencing, respectively. Pyrosequencing showed that the cheese microbiota changed dramatically during cheese processing, including during the pasteurization, starter culture, and ripening stages. Our results indicated that the RNA contents per 200 mg/200 μl of the sample increased significantly during cheese-making and ripening. The inner cheese fractions had higher RNA contents than the surfaces after 12 and 22 days of ripening in a timedependent manner (21.9 and 13.2 times higher in the inner and surface fractions than raw milk, respectively). We performed a comparative analysis of the miRNAs in each fraction by RT-qPCR. Large amounts of miRNAs (miR-93, miR-106a, miR-130, miR-155, miR-181a, and miR- 223) correlated with immune responses and mammary glands were present in aged cheese, with the exception of miR-223, which was not present on the surface. Considerable amounts of miRNAs were also detected in whey, which is usually disposed of during the cheese-making process. Unexpectedly, there were no significant correlations between immune-related miRNAs and the microbial populations during cheese processing. Taken together, these results show that various functional miRNAs are present in cheese during its manufacture and that they are dramatically increased in amount in ripened Camembert cheese, with differences according to depth.

상엽(桑葉) 추출물의 Adipogenesis 억제를 통한 항비만 활성 평가

Objectives : Mori Follium (Morus alba L. leaf) has been cultivated in many Asian countries. Especially, mulberry leaf has been used as an anti-diabetic remedy in oriental medicine. However, anti-obesity effect of mulberry has not been unknown. In this study, our objectives of study is to investigate the anti-adipogenic effect of mulberry water extract (MLE) and to reveal potential molecular anti-obesity mechanism in 3T3-L1 adipocytes differentiation model.Methods : The cytotoxicity of MLE in 3T3-L1 was examined by MTT assay. Anti-adipogenic effect of MLE was evaluated by Oil Red O (ORO) staining. To elucidate the molecular mechanism, inhibitor assay was employed. The mRNA expression levels of adipogenic transcriptional factors such as PPARγ and fatty acid synthase (FAS) were analyzed by reverse transcription-polymer chain reaction (RT-PCR) analysis.Results : The MLE treatment for 24 h did not affect to the 3T3-L1 cells at concentrations of 1, 10, 100, 200, 400, 800 and 1,000 ㎍/㎖. Thus, non-toxic concentration rages of MLE were used during adipogenesis period (day -2 to 7). Intracellular lipid accumulation in MLE-treated 3T3-L1 adipocytes (day 6) were quantitatively evaluated by ORO staining. The MLE treatment significantly and dose-dependently suppressed 3T3-L1 adipogenesis by 60.42%, 38.24%, and 5.97% at 10, 100, and 200 ㎍/㎖, respectively. In addition, our inhibitor assay and RT-PCR analysis revealed that the MLE-inhibited 3T3-L1 adipogenesis through inhibition of PPARγ mediated by Wnt/β-catenin signaling pathway.Conclusions : In conclusion, these findings indicate that the MLE could be used in prevent and/or treatment of obesity-related diseases.

상엽(桑葉) 추출물의 미백활성 및 피부섬유구조 형성에 미치는 효과

Objectives : The Skin is composed of multiple layers, including the epidermis, dermis, and hypodermis. It provides a vital barrier structure that protects vertebrates from external environmental antigens, solvents, ultraviolet light, microorganisms, toxins, and weather conditions. Although several biological effects of Mori Follium have been reported, beneficial effects of Mori Follium in skin health remain unclear. In this study, we prepared water extract of Mori Follium (MLE) and evaluated the effects on melanin accumulation and expression levels of skin fibril-related proteins.Methods : The cytotoxicities of MLE in B16F10 melanoma and human skin fibroblasts (HSF) were examined by MTT assay. Inhibitory effect of MLE on the α-MSH- and IBMX-induced melanosis in B16F10 melanoma was examined. The expression levels of fibronectin, collagen 1α2, and CCN2 in MLE-treated HSF were analyzed by reverse transcription-polymer chain reaction (RT-PCR) and western blotting.Results : The MLE treatment for 24 h did not affect to the B16F10 and HSF at concentrations of 1, 10, 50, 100, 200, 400 and 800 ㎍/ml. The MLE treatment for 72 h significantly and dose dependently suppressed melanin accumulation in B16F10 melanoma. In addition, the MLE treatment up-regulated expression levels of skin fibril-related genes such as fibronectin, collagen 1α2, and CCN2 in HSF. Our western blot analysis revealed MLE-induced up-regulation of skin fibril-related genes required the activation of CCN2 protein.Conclusions : In conclusion, these findings suggest that the MLE could be used in development of cosmetic natural material of maintaining healthy skin.

Transcriptional profiles of Rhizobium vitis-inoculated and salicylic acid-treated ‘Tamnara’ grapevines based on microarray analysis

The transcriptional profiles of ``Tamnara`` grapevine (Vitis labruscana L.) to Rhizobium vitis were determined using 12,000 gene oligonucleotide microarray chips constructed with 6,776 unigenes based on the EST sequencing. Among them, 95 clones were up-regulated more than three times and 90 were down-regulated more than 5-times in the R. vitis-inoculated grapevines relative to the control vines. Treatment of salicylic acid showed that 337 clones were upregulated and 52 clones were down regulated in grapevines. Microarray analysis, reverse transcription-polymer chain reaction, and slot blot hybridization analysis revealed that 5, 14, and 64 clones were up-regulated and 10, 12, and 61 clones were down-regulated in wounded, salicylic acidtreated, and R. vitis-inoculated ``Tamnara`` grapevine leaves, respectively. The expression patterns of β-1,3-glucanase, proline-rich protein, and lipoxygenase genes of ``Tamnara`` moderately resistant to R. vitis were similar to those of resistant ``Concord`` and ``Delaware`` grapevines. However, chalcone synthase genes in ``Tamnara`` grapevines showed similar expression patterns to susceptible grapevines ``Neomuscat`` and ``Rizamat``. Further expression studies with various clones for each gene should be conducted to elucidate their roles in resistant responses against pathogens or other stimuli in grapevines. These results could provide better resources for understanding the mechanism of defense responses against crown gall disease and clues for identifying new genes that may play a role in defense against R. vitis in grapevines.

Evaluation of Water Extract Prepared from &lt;i&gt;Chrysanthemum indicum&lt;/i&gt; Linne as Nutri-cosmetic and Cosmetic Material &lt;i&gt;In Vitro&lt;/i&gt; Model

Purpose: Chrysanthemum indicum Linne (CI) has been used as a traditional medicine. Although several biological effects of CI have been reported, the precise role and function of CI as nutri-cosmetic and cosmetic materials remain unclear. Therefore, we prepared water extract of Chrysanthemum indicum Linne (CIW). And nutri-cosmetic potential was discussed through evaluation of anti-oxidant, anti- adipogenic in 3T3-L1 cells and regulatory function of skin fibril- related genes in human skin fibroblast (HSF). Methods: The anti- oxidant activity of CIW was evaluated through metal chelating activity and reduction potential. The cytotoxicity of CIW in 3T3-L1 and HSF was evaluated by MTT assay. The anti-adipogenic effect of CIW was examined by Oil Red O (ORO) staining and microscopy observation in 3T3-L1 cells. The mRNA expression levels of skin fibril-related genes in HSF were analyzed by reverse transcription-polymer chain reaction (RT-PCR). Results: The anti-oxidant activity of CIW was increased in a dose dependent manner. The CIW treatment up to 100 µg/mL for 24 h did not affect to the viability of 3T3-L1 cells and HSF. Thus, up to 100 µg/mL of CIW was chosen in cell-based assay. The treatment of 10, 50, and 100 µg/mL CIW significantly inhibited 3T3- L1 adipogenesis by 5.17%, 72.80%, and 104.09%. In addition, up- regulated mRNA expression levels of skin fibril-related genes such as fibronectin and type Ⅰ collagen α2 (COL1A2) were observed in CIW- treated HSF. Conclusion: These results suggest that CIW has the potential as nutri-cosmetic and cosmetic ingredient which possess anti-oxidant and anti-adipogenic effects.

Expression analysis of the insulin-like growth factors I and II during embryonic and early larval development of turbot (Scophthalmus maximus)

The insulin-like growth factors I and II (IGF-I and IGF-II) are important proteins involved in fish growth and development. Here, we report the isolation of IGF-II and expression analysis of IGFs in turbot Scophthalmus maximus, aiming to clarify their function in embryonic and larval development of fish. The deduced IGF-II gene is 808 bp in full length, which encodes a protein of 219 amino acids and is 93% similar with that of Paralichthys olicaceus in amino acid sequence. The tissue abundance and the expression pattern of IGFs in a turbot at early development stages were investigated via reverse transcription-polymer chain reaction. Result showed that the IGF-I and IGF-II genes were widely expressed in tissues of S. maximus. IGF-I was detected in all tissues except intestines with the highest level in liver, while IGF-II transcript presented in all tissues except muscle. At the stages of embryonic and larval development, the mRNA levels of IGFs sharply increased from the stage of unfertilized egg to post larva, followed by a decrease with larval development. However, there was an increase in IGF-I at the embryonic stage and IGF-II at the gastrula stage, respectively. These results suggested that IGFs play important roles in cell growth and division of the turbot. Our study provides reference data for further investigation of growth regulation in turbot, which can guarantee better understanding of the physiological role that IGFs play in fish.

Detection of bovine corona virus in some governorates of Iraq

In the present study, we used Reverse transcription polymer chain reaction (RT-PCR) assay for detecting of bovine coronavirus, We evaluated the presence of bovine coronavirus ( BCoV) in diarrheic fecal samples in age (1 – 30) day. (152) fecal samples from diarrheic calves were collected from four Iraqi governorates (Al-Qadisyiah, Babylon, Wassit, and Najaf) .10 (6.57%) out of 152 were positive to bovine coronavirus. This study is the first detection of bovine corona antigen in Iraq.T he results suggest that RT-PCR is more sensitive than another method to detect BCoV, especially in subclinical cases and Because these animals shed a low amount of virus in feces and more animal show clinically normal we need to apply sensitive techniques, such as RT-PCR.

Electrospun nanofiber‐based regeneration of cartilage enhanced by mesenchymal stem cells

Application of biomaterials in combination with stem cells is a novel tissue engineering approach to regenerate cartilage. The objective of this study was to investigate the potential of poly(vinyl alcohol)/polycaprolactone (PVA/PCL) nanofiber scaffolds seeded with rabbit bone marrow-mesenchymal stem cell (BM-MSC) for cartilage tissue engineering in vitro and in vivo. We tested the biocompatibility and mechanical properties of nanofibrous scaffolds using scanning electron microscope, MTT assay, and tensile measurements. The capacity of MSC for chondrogenic differentiation on scaffolds was examined using reverse transcription-polymer chain reaction and immunostaining. For in vivo assessments, PVA/PCL nanofiber scaffolds with or without MSC were implanted into rabbit full-thickness cartilage defects. To evaluate cartilage regeneration, semi-quantitative grading and histological analysis were performed. Our results showed that PVA/PCL scaffolds supported the proliferation and chondrogenic differentiation of MSC in vitro. Moreover, the animals treated with cell-seeded PVA/PCL scaffolds showed improved healing of defects compared with untreated control and those which received cell-free scaffolds. Our findings suggest that PVA/PCL scaffolds incorporated with MSC can serve as a suitable graft for articular cartilage reconstruction.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia

Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

Downregulation of SOX7 by DNA Methylation and Its Tumor Suppressing Role In Acute Myeloid Leukemia

AbstractAbstract 4189 Background:The SOX (Sry-related HMG box) gene family is a group of transcription factors containing in common a High-Mobility-Group (HMG) box domain which shares more than 60% homology to that in Sry. SOX proteins are involved in diverse embryonic processes and recently Sox7 was shown to regulate hematopoietic stem and progenitor cells during mouse development. In this study, we examined the expression, regulation and function of SOX7 in human acute myeloid leukemia with a view to understand its link to leukemogenesis. Method:Bone marrow (BM) or blood samples from patients with primary hematological malignancies, as well as cord blood obtained from normal Caesarian Sections were prospectively collected and mononuclear cells (MNC) fractions were obtained. Screening for SOX gene expression was performed by reverse-transcription polymer chain reaction (RT-PCR) and SOX7 expression in different experiments was further evaluated by quantitative real-time RT-PCR. Methylation of CpG islands around the sox7 transcription start site was studied by bisulphate DNA sequencing and methylation-specific PCR. Leukemic cell-lines (KG1, ML2, K562) and primary AML samples were treated with demethylating agent 5-aza-2′-deoxycytidine (5AdC). Cell proliferation of GFP or GFP-SOX7 expressing K562 cells was evaluated by SNARF-1 staining, cell-cycle analysis, 3H-thymidine incorporation and clonogenic assays. Apoptosis were evaluated by Annexin V/7-AAD assay. Canonical wnt activity of K562 cells expressing GFP or GFP-SOX7 was measured by TOP-FLASH dual luciferase assay. Result:The expression of 19 SOX genes was tested by RT-PCR in normal umbilical cord blood (UCB) as well as bone marrow or blood samples from patients with hematological malignancies. SOX7 was uniquely expressed in CD34+ cells from UCB (N=11) and most case of precursor B-cell acute lymphoblastic leukemia (ALL) (17 out of 20 tested) and a ALL derived cell line Nalm-20, but not any case of acute myeloid leukemia (N=22), myelodysplastic syndrome (N=16) or chronic myelogenous leukemia (N=13). In myeloid leukemia cell lines (KG1, ML2, K562) and primary AML samples, but not Nalm-20, the transcription start site of SOX7 contained CpG islands which were heavily methylated. Treating myeloid leukemic cell lines with 5AdC induced SOX7 expression. Enforced expression of SOX7 in K562 cells by lentiviral transduction significantly reduced cell proliferation as shown by cell growth in cultures (SOX7: 6.5-fold increase; GFP: 21.7-fold increase on Day 9, N=2), SNARF-1 staining (SOX7: 57.5%; GFP: 78.0% of total cells divided twice, N=2), 3H-thymidine incorporation assay (SOX7: 3987 cpm; GFP: 5767 cpm, N=2) and colony-forming unit (SOX7: 262±99 per 1000 input cells; GFP: 464±145 per 100 input cells, p=0.055). It also induced cell cycle delay in S/G2/M phases (SOX7: 53.4±0.35%; GFP: 44.4±2.28%, p=0.029). Apoptosis was not affected. SOX7 expression in K562 cells significantly reduced canonical-wnt activity as measured by TOP-FLASH dual luciferase assay (SOX7: 30.0±7.1-fold to FOP-FLASH; GFP: 130±18.8-fold to FOP-FLASH, p=0.0081). Conclusion:SOX7 expression in AML was regulated by promoter hypermethylation and its forced expression in K562 cells reduced cell proliferation and inhibited the canonical wnt signaling pathway. The pathogenetic link between SOX7 gene silencing and AML leukemogenesis is being investigated in our laboratory. AcknowledgmentsThe project was supported by a grant from the strategic Research Theme of cancer stem cells in the HKU. Disclosures:No relevant conflicts of interest to declare.

Effects of glutamine added to enteral nutrition on Peyer's patch apoptosis in severely burned mice

Background and aims This study aimed to investigate the influence of enteral glutamine (GLN) supplementation on Peyer's patch apoptosis in severely burned mice. Methods Thirty-four mice were randomly assigned to a normal group ( n = 10), an EN group ( n = 12) and an EN supplemented with GLN (EN + GLN) group ( n = 12) and the mice in the EN and EN + GLN groups received a full-thickness scald burn over 20% total body surface area (TBSA) on the back. The burned mice then were fed orally with a common EN or an isonitrogenous and isocaloric EN supplemented with GLN for 7 days. On day 7 after injury, all surviving mice were euthanised and the entire intestine was collected. The percentage of apoptotic cells and cell percentage of phenotype in Peyer's patches were determined by flow cytometry (FCM). The FasL expression in Peyer's patches was analysed by reverse transcription polymer chain reaction (RT-PCR) and FCM. Both TNF-α levels and caspase-3 activity in Peyer's patches were also assessed. Results The results revealed that the percentage of lymphocyte subsets in Peyer's patches after burn injury significantly altered: the percentage of CD4 and CD19 cells declined and the percentage of CD8 cells correspondingly increased, when compared with the normal control mice ( p < 0.05). On the other hand, the total apoptotic ratio and all lymphocytes subset apoptosis in Peyer's patches were markedly increased ( p < 0.05), which were consistent with up-regulation in the FasL expression at the levels of both mRNA and protein, TNF-α levels and caspase-3 activity in Peyer's patches. Enteral GLN supplementation partially reversed these changes: the total apoptotic ratio and all lymphocytes subpopulation apoptosis in Peyer's patches were markedly decreased when compared with the EN group ( p < 0.05). The percentage of lymphocyte subsets within Peyer's patches also restored the condition prior to injury. However, no significant differences in the FasL expression, including mRNA and protein, were observed between the EN and EN + GLN groups. Although, both TNF-α levels and caspase-3 activity in Peyer's patches were lower in the EN + GLN group than in the EN group ( p < 0.05). Conclusions This study suggests that enteral GLN supplementation is superior to a common enteral nutrition with respect to attenuating apoptosis in Peyer's patches, which might be more effective in decreasing TNF-α levels and down-regulating caspase-3 activity in Peyer's patches.

Bacille‐Calmette Guèrin induces caspase‐independent cell death in urothelial carcinoma cells together with release of the necrosis‐associated chemokine high molecular group box protein 1

To evaluate the ability of bacille-Calmette Guèrin (BCG) to induce caspase-independent cell death and release the necrosis-associated chemokine high molecular group box protein 1 (HMGB1) from urothelial carcinoma (UC) cells; a correlative clinical trial determined if BCG treatment resulted in increased urinary levels of HMGB1. The human UC cell lines 253 J and T24 were pretreated with apoptosis inhibitors, exposed to BCG, and cell viability and ultrastructural changes measured. HMGB1 levels were assessed in cell culture supernatant after BCG treatment. The expression/function of HMGB1 receptors on the UC cell lines was determined by reverse transcription-polymer chain reaction and the ability of exogenous HMGB1 to activate nuclear factor (NF)-kappaB signalling assessed. An HMGB1 enzyme-linked immunosorbent assay was used to measure HMGB1 levels in urine obtained from BCG-treated patients. Inhibition of apoptotic pathways failed to inhibit BCG-induced cell death in UC cells. Electron microscopy showed BCG-dependent ultrastructural changes consistent with cellular necrosis. BCG exposure resulted in a binary increase in cell culture supernatant levels of HMGB1. UCs expressed multiple HMGB1 receptors. Treatment of UCs with HMGB1 activated NF-kappaB. In the clinical setting, six of seven patients had increased urinary levels of HMGB1 at 24 h after BCG treatment. BCG causes direct cytotoxicity in a subpopulation of UC cells. This cytotoxicity is caspase-independent and associated with ultrastructural changes and cellular protein release (HMGB1), characteristic of necrosis. Urinary levels of HMGB1 can be elevated in patients after BCG treatment. The expression and function of HMGB1 receptors in UC cells, coupled with the known role of HMGB1 on the host immune response, suggest a role for necrosis and HMGB1 release in the antitumour effect of BCG.

Inducible Nitric Oxide Synthase Expression in the Intestinal Muscularis Mediates Severe Smooth Muscle Dysfunction During Acute Rejection in Allogenic Rodent Small Bowel Transplantation

Acute rejection in small bowel transplantation is associated with dysmotility. Therefore, host and organ not only face the threat of destructive immunological processes but also the risk of bacterial translocation, endotoxemia, and systemic inflammatory response syndrome. We hypothesized that dysmotility during acute rejection is based on an alloreactive leukocyte infiltrate and coexpression of the kinetically active mediator inducible nitric oxide synthase (iNOS) in the muscularis propria. Allogenic and isogenic rat small bowel transplantation (SBTx; Brown Norway [BN] to Lewis and BN to BN) was performed without immunosuppression. Animals were sacrificed 4 and 7 d after SBTx. Leukocyte infiltration and iNOS protein was investigated by immunohistochemistry and immunohistology. Real-time reverse transcription polymer chain reaction was used to detect iNOS expression. Griess reaction was used to evaluate NO production. Spontaneous, bethanechol-stimulated, and L-N(6)-(1-iminoethyl)-L-Lysin-blocked jejunal circular muscle contractions were measured in a standard organ bath in vitro. On d 7 after SBTx, allogenic transplanted animals showed significant infiltration with ED-1- and ED-2-positive monocytes and macrophages within the muscularis parallel to the manifestation of acute rejection. Additionally, immunohistochemistry localized iNOS protein in leukocytes within the muscularis. Reverse transcription polymer chain reaction showed a significant increase in iNOS mRNA expression (460-fold) in allogenic transplanted muscularis compared to isogenic transplanted muscularis (2.5-fold). Compared to controls, allogenic grafts showed a 73% decrease in smooth muscle contractility, while isogenic grafts showed only an 8% decrease of contractility on d 7. L-N(6)-(1-iminoethyl)-L-Lysin application in vitro significantly improved muscle contractility and decreased NO production. The data show that inflammation associated iNOS expression in the intestinal graft muscularis is involved in motoric graft dysfunction during acute rejection.

Analysis by cDNA microarrays of altered gene expression in middle ears of rats following pneumococcal infection

Objective: Streptococcus pneumoniae is the most common pathogen in otitis media. Infection of the middle ear with S. pneumoniae potentiates development of thick effusion in the middle ear which frequently causes hearing loss and communication disorders in children. What has changed immediately in the middle ear cleft following pneumococcal infection is extensively studied and characterized but what has changed ever after remains elusive. The purpose of this study is to explore the cellular and molecular basis that remains on a longer time after acute pneucmococcal middle ear infection and potentiates development of thick effusion in the middle ear. Methods: 12 rats were intrabullarly inoculated with pneumococcus at 2.5×10 6 CFU/ear and profiles of gene expression in the middle ear were examined by cDNA microarrays in combination with reverse transcription-polymer chain reaction (RT-PCR) 6 weeks after infection while the morphologic changes in middle ear were simultaneously characterized by histopathologic techniques. Twelve rats receiving phosphate-buffered saline (PBS) served as controls. Results: it demonstrated that pneumococcus infected ears had the expression of the following genes at a high level compared to the controls: mitogenic signaling proteins (mitogen-activated protein kinase [MEK1 and MEK2], helix–loop–helix transcriptional regulators (Id3 and Id1), ion channels (sodium channel beta 1 and sodium channel 2), and mucin glycoproteins ( Muc2 and Muc5). The morphology demonstrated a thickened mucosa and submucosa with increased expression of macroglycoconjugates compared to the controls. Conclusion: the expression of several genes remains high even after the acute episode of pneumococcal otitis media has been resolved. The up-regulated expression of these genes may serve as the basis for the development of thick effusion and mucous cell metaplasia/hyperplasia once it is complicated with other factors such as dysfunction of the Eustachian tube.