Enzyme-linked Immunosorbent Assay Results Research Articles

Impact of Fermented Corn-Soybean Meal on Gene Expression of Immunity in the Blood, Level of Secretory Immunoglobulin A, and Mucosa-Associated Bacterial Community in the Intestine of Grower-Finisher Pigs.

This study was conducted to determine the effect of a fermented corn–soybean meal [fermented feed (FF)] on the gene expression of immunity in the blood, the level of secretory immunoglobulin A (sIgA), and mucosa-associated bacterial community in the duodenum and colon of grower-finisher pigs. In this study, crossbred barrows (Duroc × Landrace × Large White) were randomly assigned to either an unfermented corn–soybean diet (Ctrl) (n = 6) or an FF diet (n = 6), and then the following were examined: the expression of immunity using real-time reverse transcription polymerase-chain reaction in the blood, sIgA using enzyme-linked immunosorbent assay (ELISA), and changes in the bacterial community using Illumina Hiseq sequencing in the mucosa of the duodenum and colon. Compared with control pigs fed with a standard diet, the results showed that FF caused upregulation of the mRNA expression of Toll-like receptor 3 (TLR3), TLR4, TLR6, and TLR8 in the blood (P < 0.05). Moreover, sequencing of 16S rRNA genes in duodenal mucosa samples indicated that the FF diet had a lower proportion of Tenericutes (P < 0.05) in the duodenal mucosa-associated microbiota, and FF significantly increased the percentage of Rikenellaceae and Christensenellaceae but decreased the abundance of Lachnospiraceae (P < 0.05) in the colonic mucosa-associated microbiota. The ELISA results showed that FF significantly increased the concentration of sIgA in the colonic mucosa (P < 0.05). More importantly, our correlation analysis indicated that the gene expression of immunity in the blood and the concentration of sIgA was associated with colonic mucosa-associated microbiota. Our data provide new knowledge into the adaptation response of the intestine to fermented feeding in monogastric animals.

1719-P: Novel Mass Spectrometry–Based Selected Reaction Monitoring Proteomics for Analysis of Low Abundant Insulin Levels in Cerebrospinal Fluid

Circulating insulin levels are routinely measured using Enzyme-linked Immunosorbent Assay (ELISA). However, ELISA is not sensitive enough to measure cerebrospinal fluid (CSF) insulin levels which are 10-100 times lower than those of plasma: yet ELISA results are often reported for insulin analysis in CSF where it serves as a surrogate for brain interstitial fluid insulin levels. We developed a novel LC-MS/MS based selected reaction monitoring (SRM) proteomics assay for quantitative analyses targeting predetermined peptides derived from trypsin digests of insulin; each peptide depends on specific SRM transitions. We determined insulin levels using our novel insulin SRM proteomics method in the matching plasma and CSF samples that were from individuals fasting ≥12 hours and individuals after 2 hours of steady state hyperglycemia by clamp technology (2 hr SS HG). All samples were obtained in individuals with normal fasting glucose and HbA1C levels. Statistically significant differences were observed in both plasma and CSF between fasting and 2 hr SS HG by two tailed and unpaired student t test and 2-way matched ANOVA as shown in Table below. Our results confirmed that peripheral endogenous insulin appears in CSF at least 2 hours after steady state hyperglycemia in subjects with normal glucose tolerance. Our SRM insulin tryptic peptide panel reliably quantified insulin peptide levels in human CSF samples. Disclosure Q. Liu: None. M. Zhu: None. C.W. Chia: None. J.F. O’Connell: None. P. Zhang: None. J.M. Egan: None. Funding National Institute on Aging

Transplantation of umbilical cord blood mononuclear cells attenuates the expression of IL-1β via the TLR4/NF-κB pathway in hypoxic-ischemic neonatal rats

Objective:This study aims to observe the effects of transplantation of umbilical cord blood mononuclear cells (UCBMCs) on the expression of interleukin (IL)-1β and explore the mechanism via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway in hypoxic-ischemic neonatal rats.Methods:Seven-day-old Sprague-Dawley neonatal rats were randomly divided into Sham, hypoxic-ischemic brain damage (HIBD), and UCBMC groups. The HIBD model was prepared by Rice-Vannucci method, and UCBMC were transplanted 24 h after HIBD in the UCBMC group. At 7 days after transplantation, changes in neurons and the TLR4 protein were examined by neuronal nuclei (NeuN)/TLR4 immunofluorescence staining. The expression of pNF-κB and IL-1β proteins was detected by immunohistochemical staining and enzyme linked immunosorbent assay (ELISA).Results:The percentage of NeuN+DAPI+ cells in the injured cortex in the UCBMC group was significantly higher than that in the HIBD group and lower than that in the Sham group (P &lt; 0.05). The number of NeuN+TLR4+DAPI+cells in the UCBMC group was significantly lower than that in the HIBD group (P &lt; 0.05) but higher than that in the Sham group (P &lt; 0.05). More pNF-κB+ cells were observed in the HIBD group than in Sham and UCBMC groups (P &lt; 0.05), and more pNF-κB+ cells were observed in the UCBMC group than in the Sham group (P &lt; 0.05). ELISA results showed that the IL-1β expression in the injured cerebral cortex in the UCMBC group was significantly lower than that in the HIBD group but remained higher than that in the Sham group (P &lt; 0.05).Conclusions:UCBMC transplantation could inhibit the IL-1β protein expression in the injured cortex, thereby alleviating HIBD in neonatal rats. The underlying mechanism might be associated with the down- regulation of TLR4 and pNF-κB proteins.

MiR-199a modulates autophagy and inflammation in rats with cerebral infarction via regulating mTOR expression.

The aim of this study was to investigate the roles of micro ribonucleic acid (miR)-199a in rats with cerebral infarction by regulating mammalian target of rapamycin (mTOR). A total of 36 Sprague-Dawley rats were randomly assigned into three groups, including: sham group (n=12), model group (n=12) and miR-199a mimics group (n=12). In sham group internal and external carotid arteries were exposed. The ischemia-reperfusion model was successfully established using suture embolization in the other two groups. After modeling, rats in sham group and model group were intraperitoneally injected with normal saline. However, rats in miR-199a mimics group were injected with miR-199a mimics. Following intervention for 3 d, sampling was conducted. Neurological deficit was evaluated in rats based on the Zea-Longa scoring system. Hematoxylin-eosin (HE) staining was performed to observe neuronal morphology. The expression of mTOR was detected using immunohistochemistry, and the relative expression level of tau protein was determined via Western blotting (WB). Besides, the messenger RNA (mRNA) expressions of mTOR and tau were detected by quantitative Polymerase Chain Reaction (qPCR). Finally, inflammatory factor content was measured through enzyme-linked immunosorbent assay (ELISA). Model group and miR-199a mimics group exhibited a substantially higher Zea-Longa score than sham group (p<0.05). Compared with model group, the Zea-Longa score rose prominently in miR-199a mimics group (p<0.05). According to the results of HE staining, the structure of neurons in sham group was clear and intact, while the structure of neurons in model group was disordered. Meanwhile, neuronal morphology in miR-199a mimics group was significantly worse than that in model group (p<0.05). Immunohistochemistry results demonstrated that the positive expression level of mTOR was considerably upregulated in both model group and miR-199a mimics group in comparison with sham group (p<0.05). Moreover, its positive expression level in miR-199a mimics group was markedly higher that in model group (p<0.05). Based on the results of WB, model and miR-199a mimics groups exhibited a remarkably higher relative expression level of tau protein than sham group (p<0.05). However, the relative expression level of tau protein in miR-199a mimics group was prominently higher than that in model group (p<0.05). QPCR results manifested that the relative mRNA expression levels of mTOR and tau in model group and miR-199a mimics group were dramatically higher than those in sham group (p<0.05). Compared with those in model group, the relative mRNA expression levels of mTOR and tau increased significantly in miR-199a mimics group (p<0.05). ELISA results revealed that model group and miR-199a mimics group had prominently higher content of inflammatory factors than sham group (p<0.05). In addition, content of inflammatory factors in miR-199a mimics group was considerably higher than that in model group (p<0.05). MiR-199a modulates mTOR expression to exert important regulatory effects on the autophagy and inflammation in rats with cerebral infarction.

A Pilot Comparative Study Between Serological and Genetic Investigations in Relationship to Clinical Outcomes on Patients with Cystic Echinococcosis.

SummaryThe aim of this study was to investigate whether Enzyme-Linked Immunosorbent Assays (ELISA) and Western Blotting (WB) methods could contribute to the assessment of clinical outcomes in genotype-defined cystic echinococcosis (CE) patients. Twenty-nine human isolates and blood samples have been taken from patients who underwent surgery or percutaneous aspiration (PAIR) for therapeutic purposes at Ege University and Manisa Celal Bayar University Hospitals. All sera of patients were screened for the presence of E. granulosus IgG antibodies using in-house approved ELISA and WB methods. According to the ELISA results, five patients had high, thirteen patients had medium and eight patients had low specific antibody level response which ranged 1/640 -1/5000. Despite confirmed WB positivity three patients were found to be negative by ELISA. Immunoblot analysis of EgAg showed many protein bands with size of 8, 12, 20, 22, 24, 36, 75 and 90 kDa. Among of them, 8 – 12 kDa bands (90 %), 20 – 22 kDa and 36 kDa bands presented strong reactivity against human serum specimens. No serum samples from healthy control reacted with EgAg. Phylogenetic analysis of resulting COX1 and NAD1sequences has revealed that all patients in our study were infected with the E. granulosus G1-G3 genotype. There was no consistent correlation between results of ELISA and WB, the number or size of cysts and genotype. Our study brings a unique contribution in terms of relationship between serological investigation, disease genotypes and clinical outcomes.

Clinical Significance of M1/M2 Macrophages and Related Cytokines in Patients with Spinal Tuberculosis

Background Macrophages are important immune cells involved in Mycobacterium tuberculosis (M.tb) infection. To further investigate the degree of disease development in patients with spinal tuberculosis (TB), we conducted research on macrophage polarization. Methods Thirty-six patients with spinal TB and twenty-five healthy controls were enrolled in this study. The specific morphology of tuberculous granuloma in spinal tissue was observed by hematoxylin-eosin (H&E) staining. The presence and distribution of bacilli were observed by Ziehl-Neelsen (ZN) staining. Macrophage-specific molecule CD68 was detected by immunohistochemistry (IHC). M1 macrophages play a proinflammatory role, including the specific molecule nitric oxide synthase (iNOS) and the related cytokine tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). M2 macrophages exert anti-inflammatory effects, including the specific molecule CD163 and related cytokine interleukin-10 (IL-10). The above markers were all detected by quantitative real-time PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and IHC. Results Typical tuberculous granuloma was observed in the HE staining of patients with spinal TB. ZN staining showed positive expression of Ag85B around the caseous necrosis tissue and Langerhans multinucleated giant cells. At the same time, IHC results indicated that CD68, iNOS, CD163, IL-10, TNF-α, and IFN-γ were expressed around the tuberculous granuloma, and their levels were obviously higher in close tissue than in the distant tissue. RT-PCR and ELISA results indicated that IL-10, TNF-α, and IFN-γ levels of TB patients were also higher than those of the healthy controls. Conclusion The report here highlights that two types of macrophage polarization (M1 and M2) are present in the tissues and peripheral blood of patients with spinal TB. Macrophages also play proinflammatory and anti-inflammatory roles. Macrophage polarization is involved in spinal TB infection.

Development and Validation of KASP Markers for the Identification of Pea seedborne mosaic virus Pathotype P1 Resistance in Pisum sativum.

As pesticides have become heavily relied on for management of insect pests vectoring economically important pathogens of vegetable crops, development of pathogen-resistant germplasm remains a promising alternative to reduce or eliminate costly and timely chemical inputs. Molecular markers can be used to rapidly identify resistant genotypes to aid breeders in advancing germplasm. This study developed two kompetitive allele-specific PCR (KASP) genotyping markers for rapid screening of Pisum sativum genotypes for resistance to Pea seedborne mosaic virus pathotype P1 (PSbMV-P1), the most economically devastating strain worldwide. The KASP markers differentiate two eIF4E PSbMV-P1-resistant allelic variants from susceptible eIF4E variants. A single nucleotide polymorphism (Resistant 1) and a 3-basepair deletion (Resistant 2) present in either of the two resistant alleles were used for marker design. Forty-four P. sativum lines previously characterized for resistance to PSbMV were inoculated with PSbMV-P1 in a greenhouse, observed for visual symptoms, assayed for virus susceptibility by enzyme-linked immunosorbent assay (ELISA), and genotyped by KASP marker analysis. The KASP markers were 100% accurate in characterizing PSbMV-P1-susceptible and PSbMV-P1-resistant genotypes when correlated with the ELISA results. The Resistant 1 marker also correlated with resistance to PSbMV pathotypes P2 and P4 completely, making this marker a new advanced tool for P. sativum breeding programs.

The effect of cytoplasmic crude extracts of Trichophyton verrucosum on cell mediated immunity

ObjectiveTrichophyton verrucosum is a slow growing dermatophyte responsible for a number of skin diseases such as ringworm, and is characterized by patches of hair loss and thick crusts on the host skin in domestic animals. In this study, we examined the immunomodulatory effects of crude extract of Trichophyton verrucosum (TV)cytoplasm in a mouse model. MethodsThe TV variate was cultured on Sabouraud dextrose agar and the mycelium was grinded by mechanical force. The purified protein was obtained from crude extract of the fungus, and protein concentration was measured by BradFord assay. Six to eight week-female BALB/c mice were divided into three groups: test group, receiving cytoplasmic crude extract plus defibrinated sheep blood; control group, receiving defibrinated sheep blood; and normal group, receiving normal saline. Injections were performed on days 0, 3, 5, 7 and 9 and the mice were sacrificed four days after the last injection. T lymphocyte metabolic activity was examined by methyl thiazol tetrazolium (MTT) assay, and also interleukin-4 (IL-4) and interferon-γ (IFNγ) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). ResultsMTT assay showed that the TV extract stimulated lymphocyte metabolic activity. ELISA results showed that despite increase in the level of IFNγ, no changes were observed in IL-4 level. ConclusionsResults indicated that crude extract of TV cytoplasm may probably act as an immune modulator, which affects Th1 responses. The TV crude extract may be an appropriate agent to induce cellular immunity for combating dermatophytosis infection in animals; and therefore, TV extract may have some potential applications in vaccine/adjuvant technology.

Identification of an Indiscriminate Peptide by Phage Display Technology

Phage display has been widely used to develop high‐affinity peptide ligands against proteins and cell lines. Such peptides are typically selected using biopanning to exhibit significant binding affinity and specificity against a molecular target. Rarely are the same peptides identified from separate phage display selections, since the presence of these ligands in a selection typically signify specific binding to the target. However, here, we report the identification of the same peptide (TG1) from two different phage display selections against the hemagglutinin (HA) epitope and human pancreatic cancer (Mia Paca‐2) cells.Phage display selections were conducted to identify a peptide with high affinity and specificity for the HA epitope. In brief, a pre‐cleared 15‐mer fUSE5 phage library was subjected to four rounds of selections against 0.04–4 μg HA. Following the last round of selection, the phage DNA was amplified using PCR and submitted to next‐generation sequencing. These results identified peptide TG1 (RNVPPIFNDVYWIAF) as the most prevalent ligand in the selection. Modified enzyme‐linked immunosorbent assay (ELISA) results showed that 109 nM TG1 bound significantly higher (p&lt;0.05) to 5 μg immobilized HA compared to a non‐relevant peptide J18 known to bind ovarian adenocarcinoma (SKOV‐3) cells. In a separate, previously reported phage display selection, the same 15‐mer fUSE5 phage library was subjected to selections against human pancreatic cancer (Mia‐Paca‐2) cells. Again, next‐generation sequencing results identified TG1 as the third most prevalent peptide. Modified ELISA results using 10 μM TG1, and a known pancreatic cancer‐targeting peptide MCA2, showed that both ligands bound significantly (p&lt;0.01) to Mia‐Paca‐2 cells compared to dimethyl sulfoxide (DMSO; vehicle). A literature search revealed that TG1 was identified by two separate research groups conducting phage display selections against caveolin protein and mouse embryos. These results indicate that the peptide is an indiscriminate binder. Thus, we sought to elucidate the binding properties of peptide TG1 and phage TG1 (pTG1).Modified ELISA with 10 μM TG1, MCA2, J18, or DMSO were carried out against SKOV‐3 and human normal pancreatic (hTERT‐HPNE) cells. Results showed that TG1 bound significantly higher (p&lt;0.01) to both SKOV‐3 and hTERT‐HPNE cells compared to DMSO. As expected MCA2 showed no binding to either cell line. Peptide J18 showed significant binding (p&lt;0.05) to SKOV‐3 cells.Phage qPCR analysis was performed to further elucidate the binding properties of the pTG1. 1010 V/mL pTG1, pancreatic cancer‐targeting pMCA1, or WT phage was incubated with Mia‐Paca‐2, SKOV‐3, or hTERT‐HPNE cells. After washing with Tris‐buffered saline (TBS), bound phage were eluted with 2.5% 3‐cholamidopropyl dimethylammonio 1‐propanesulfonate (CHAPS). Collected phage virions (5 μL) were quantified by qPCR using Fast SYBR Green Master Mix on an Applied Biosystems StepOnePlus Real‐Time qPCR System (Applied Biosystems, CA). The results revealed that pTG1 demonstrated significantly higher binding (p&lt;0.01) compared to WT for all cell lines. As expected, pMCA1 showed binding to Mia‐Paca‐2, and pJ18 exhibited binding to SKOV‐3 cells. Taken together, these results indicate that TG1 indiscriminately binds multiple targets.

Effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells

ObjectiveTo investigate the role of clindamycin hydrochloride on reversing Lipopolysaccharide(LPS)‐induced proliferation inhibition and Tumor necrosis factor‐α(TNF‐α) secretion in human periodontal ligament cells.MethodHuman periodontal ligament cells (HPDLCs) was cultured in vitro and identified by immunohistochemical staining. 4–8 generations HPDLCs were selected for the experiment. Above HPDLCs were divided into control group, LPS group and drug group. In the control group, only Dulbecco's Modified Eagle Medium(DMEM) culture solution was added to each hole. In the LPS group,100 μg/ml LPS was added to DMEM culture solution for each hole.In the drug group, different concentrations(10μg/ml,40μg/ml and 160μg/ml) of clindamycin hydrochloride and 100μg/ml LPS were distributed to different test Wells.MTT and enzymelinkedimmunosorbentassay(ELISA) were used to detect the effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells.ResultsMTT results showed that the absorbance of MTT in the LPS group decreased significantly compared with that in the control group. Compared with LPS group, the absorbance of MTT in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of increase. ELISA results showed that the level of TNF‐α in the LPS group increased significantly compared with that in the control group. Compared with LPS group, the level of TNF‐α in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of decrease.ConclusionClindamycin hydrochloride can reverse LPS‐induced proliferation inhibition, which is beneficial to the regeneration of periodontal tissue and the production of new periodontal attachment. It also can significantly inhibit the role of LPS in promoting the secretion of TNF‐α by HPDLCs, thereby reducing the inflammatory response of periodontal tissues, which is beneficial to the treatment of periodontal disease.Support or Funding InformationThis study was supported by Lvliang City Science and Technology Fundation Grant, Lvliang people‘s hospital Fundation Grant and China Postdoctoral Science Fundation Grant (2019T120195)

Anti-Inflammatory Effects of Cerium Dioxide Nanoparticles on Peritonitis in Rats Induced by Staphylococcus epidermidis Infection

Objective. To investigate the effects of cerium dioxide (CeO2) nanoparticles on the inflammatory response of peritonitis rats induced by Staphylococcus epidermidis infection. Methods. Green tea polyphenol CeO2 nanoparticles were synthesized and characterized by transmission microscopy, ultraviolet-visible spectroscopy, FT-IR, and powder diffractometer. 40 male adult SD rats were randomly divided into 4 groups (n = 10 each): a control group, a model group, a CeO2 group, and a CeO2 + model group. Staphylococcus epidermidis solution was injected intraperitoneally with 107 CFU/ml of bacterial solution in the model group, while the control group was injected intraperitoneally with the same amount of normal saline, and the CeO2 and CeO2 + model groups were injected with 0.5 mg/kg CeO2 nanoparticles through the tail vein for 2 h and then injected with saline or bacterial solution for 2 h, respectively. After 0 h, 3 h, 12 h, 24 h, and 48 h of model construction, rats were sacrificed, and serum and peritoneal lavage fluid were collected. The total number of leukocytes and the percentage of each type of leukocytes in the peritoneal lavage fluid were determined. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factor TNF-α in serum and peritoneal lavage fluid, and myeloperoxidase (MPO) activity in peritoneal tissue was also measured. In addition, real-time fluorescence quantitative PCR (RT-PCR) was used to measure the expression of TLR2 and TLR4 in peritoneal tissue, and western blotting was used to detect the expression of TLR2, TLR4, and the activation of NF-κB signaling pathways as well. Results. The CeO2 has an average size of 37 ± 3 nm with binding activity to proteins, phenolic compounds, and alkaloids. After counting the white blood cells in the peritoneal lavage fluid, it was found that the total number of white blood cells and the percentage of neutrophils in the model group were significantly increased (both P&lt;0.05), and CeO2 treatment significantly reversed the above changes (both P&lt;0.05). The ELISA results showed that compared with the control group, the TNF-α in the peritoneal lavage fluid and serum of the model group increased in a time-dependent manner (all P&lt;0.05); however, there was no significant change in the CeO2 group (P&gt;0.05); at the same time in the CeO2 + model group, the TNF-α content was significantly reduced (all P&lt;0.05). Detection of MPO activity in peritoneal tissue revealed that MPO activity was significantly increased under peritonitis (all P&lt;0.05), and CeO2 treatment could mitigate that increase (all P&lt;0.05). RT-PCR results showed that compared with the control group, the expression of TLR2 and TLR4 mRNA levels in the peritoneum of the model group were increased in a time-dependent manner (all P&lt;0.05), and there was no significant change in the CeO2 group (P&gt;0.05); however, TLR2 and TLR4 mRNA levels were significantly reduced in the CeO2 + model group (all P&lt;0.05). Western blotting test was performed on the peritoneal tissue collected after 48 h of the model establishment. Compared with the control group, the levels of TLR2, TLR4, p–NF–κB, and p-IκBα protein in the model group were significantly increased (all P&lt;0.05), while CeO2 group showed no significant changes (P&gt;0.05) and administration of CeO2 before model construction can significantly reverse the above protein activation (all P&lt;0.05). Conclusion. CeO2 nanoparticles have anti-inflammatory effects in peritonitis caused by Staphylococcus epidermidis infection.

Inhibition of nuclear factor kappa B as a mechanism of Danshensu during Toll-like receptor 2-triggered inflammation in macrophages

Danshensu (DSS) is a water-soluble phenolic compound in Danshen (Salvia Miltiorrhiza Radix et Rhizoma). Although various pharmacological activities have been recognized, little is known regarding its anti-inflammatory effect and related molecular mode of action. In the current study, bone marrow-derived macrophages (BMMs) were activated by a Toll-like receptor 2 (TLR2) agonist Pam3CSK4 with or without DSS intervention. Production of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-12 (IL-12) was detected by both enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR). Activation of signaling pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) was assessed by Western blot. Additionally, RNA sequencing (RNA-seq) combined with bioinformatics analyses was applied to investigate the molecular mechanisms of DSS. Emphasis was placed on the construction of the protein–protein interaction (PPI) network and transcription factor (TF) enrichment analysis of data including co-regulated differentially expressed genes (DEGs) in the Pam3CSK4 vs. control and DSS vs. Pam3CSK4 groups. The RT-qPCR and ELISA results showed that DSS effectively inhibited the expressions of IL-6 and IL-12, indicating a significant anti-inflammatory effect. Western blot verified that DSS suppressed the phosphorylation of p65, which was in accordance with the results of the TF enrichment analysis. Additionally, the PPI network analysis showed several key molecules, including lactoferrin (Ltf), CC-chemokine receptor 7 (Ccr7), interferon-gamma (IFN-γ) and C-X-C motif chemokine ligand 9 (Cxcl9), to be regulatory genes that responded to DSS treatment. Overall, our study revealed that DSS has a pronounced anti-inflammatory effect involving TLR2 and macrophages through the NF-κB signaling pathway, which supports the novel application of DSS in the treatment of relevant diseases including atherosclerosis and ischemic or ischemic/perfusion injury of the heart and brain.

Effect of lncRNA AK125437 on postmenopausal osteoporosis rats via MAPK pathway.

The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) AK125437 on rats with postmenopausal osteoporosis via the mitogen-activated protein kinase (MAPK) pathway. A total of 36 Sprague-Dawley rats were randomly divided into three groups, including normal group, model group, and an inhibitor group, with 12 rats in each group. Only ovaries were exposed in normal group. The postmenopausal osteoporosis model was established in model group. Meanwhile, the intervention was performed with inhibitor for 3 months after modeling in inhibitor group, followed by sampling. The expression of receptor activator of nuclear factor kappa-B ligand (RANKL) was detected via immunohistochemistry. The protein expression level of phosphorylated p38 (p-p38) MAPK was determined via Western blotting (WB). Furthermore, the expression level of lncRNA AK125437 and the content of serum estradiol were determined via quantitative Polymerase Chain Reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. In addition, bone mineral density was measured using dual-energy X-ray bone mineral absorptiometer. Immunohistochemistry results indicated that model group and inhibitor group had notably up-regulated positive expression level of RANKL than normal group (p<0.05), which was remarkably lower in inhibitor group than model group (p<0.05). Western blot results showed that compared with normal group, the protein expression level of p-p38 MAPK was substantially elevated in model and inhibitor groups (p<0.05). Meanwhile, the protein expression level of p-p38 MAPK was markedly lower in inhibitor group than that in model group (p<0.05). According to qPCR results, the expression level of lncRNA AK125437 was significantly up-regulated in both model group and inhibitor group compared with normal group, showing statistically significant differences (p<0.05). However, no significant differences were observed between model group and inhibitor group (p>0.05). ELISA results revealed that model group and inhibitor group had markedly lower estradiol content than normal group (p<0.05). There was no statistically significant difference in the content of estradiol between the two groups (p>0.05). According to the measurement results of bone mineral density, compared with normal group, bone mineral density was notably lower in model group and inhibitor group (p<0.05). Furthermore, it was markedly higher in inhibitor group than that of model group (p<0.05). LncRNA AK125437 affects the bone mineral density of rats with postmenopausal osteoporosis by activating the MAPK pathway.

Comparison of brain injuries in rat cardiac arrest models induced by asphyxia and electrical stimulation

To compare the severity of brain injury between asphyxia and electrical stimulation induced cardiac arrest in rats. Forty-two healthy male Sprague-Dawley (SD) rats were randomized into sham group (n = 6), asphyxia group (n = 18) and electrical stimulation group (n = 18). Rats in each group were given invasive mechanical ventilation and femoral blood vessels catheterization for monitoring blood pressure and fluid infusion. In the asphyxia group, the tracheal tube was clamped to induce cardiac arrest, and in the electrical stimulation group, the esophageal electrical stimulation was used to induce cardiac arrest, and cardiopulmonary resuscitation (CPR) was performed 4 minutes after cardiac arrest. In the sham group, only tracheal intubation and femoral artery intubation were performed after anesthesia, but cardiac arrest was not induced. Animals were allowed to survive until 72 hours after resuscitation, and survival analysis was performed using Kaplan-Meier curves. At 24 hours and 72 hours after resuscitation, the neurological deficit score (NDS) was measured. The vena cava blood was collected, and the brain injury associated serum biomarkers, neuron-specific enolase (NSE) and S100B, were detected by enzyme-linked immunosorbent assay (ELISA). The brain tissues were then harvested to perform hematoxylin-eosin (HE) staining for observing pathological changes in the hippocampal CA1 area with light microscopy. Cardiac arrest was successfully induced in both the asphyxia group and the electrical stimulation group, 94.4% (17/18) and 88.9% (16/18) animals were resuscitated successfully in the two groups respectively. Kaplan-Meier curves analysis showed that 72-hour cumulative survival rate was similar in the asphyxia group and the electrical stimulation group (Log-Rank test: χ2 = 0.040, P = 0.841). Both asphyxia group and electrical stimulation group had higher NDS score than sham group at 24 hours after resuscitation (37.50±4.26, 32.17±4.02 vs. 8.33±2.33, both P < 0.01). NDS score showed a downwards trend at 72 hours after resuscitation in both model groups, and the decline was more significant in the electrical stimulation group, which was significantly different as compared with asphyxia group (14.00±2.89 vs. 26.33±4.84, P < 0.05). ELISA results showed that the levels of serum NSE at 24 hours after resuscitation in the asphyxia and electrical stimulation groups were significantly higher than those in the sham group (μg/L: 1.02±0.07, 1.02±0.02 vs. 0.87±0.02, both P < 0.05). NSE kept increasing at 72 hours after resuscitation in the asphyxia group, which showed significant difference as compared with sham group (μg/L: 1.03±0.05 vs. 0.87±0.02, P < 0.01). But it had almost recovered to the normal level in the electrical stimulation group without significant difference as compared with sham group (μg/L: 0.96±0.04 vs. 0.87±0.02, P > 0.05). There was no significant difference in S100B level at different time points after resuscitation among three groups. It was displayed under light microscope that there was no significant neuronal damage in the hippocampal CA1 area in the two model groups at 24 hours after resuscitation as compared with the sham group. At 72 hours, there were certain damages in the hippocampal CA1 area in both model groups, which were more obvious in the asphyxia group. Both cardiac arrest models induced by asphyxia and electrical stimulation show a certain degree of brain injuries after resuscitation. Brain injuries are more severe in asphyxia-induced cardiac arrest compared with trans-esophageal electrical stimulation method.

Peptide specifically binding to human medullary breast carcinoma Bcap-37 cells selected by phage display in vivo and its property as well as combination effect

Objective To investigate the property and the combination effect of the peptide specifically binding to human medullary breast cancer Bcap-37 cells by using phage display in vivo and to provide molecular targeting probe for early diagnosis of breast cancer. Methods The human medullary breast carcinoma Bcap-37 cells tumor-bearing nude mice model was prepared and three rounds in vivo were performed by using Ph.D.-C7CTM phage display peptide library. The distribution of screened phages in tumors and normal tissues was detected by using immunohistochemistry. The affinity of monoclonal phage to Bcap-37 cells was identified by using enzyme linked immunosorbent assay (ELISA). The positive monoclonal phage DNA was taken and sequenced, and the sequence with high repetition rate was selected to synthesize peptide by using chemical methods. Optical molecular probe was prepared and fluorescence molecular imaging was used to test its specificity and targeting ability for breast transplantation tumor of tumor-bearing nude mice in vivo. Results The recovery rate of phage in the third round screening in vivo was 107.2 times than that in the first round. Immunohistochemical results showed that the phages binding to the tumor tissues were increased successively with the increasing screening rounds in vivo. The number of phages binding to tumor tissues was more than that binding to normal tissues (lung, skeletal muscle, liver and kidney). The absorbancy (A) value of section scanning image in the tumor tissues was higher than that in the normal tissues, and the difference was statistically significant different (P < 0.05). ELISA results showed that 22 phages (affinity≥2) among the 50 randomly selected monoclonal phage were positive. After DNA sequencing analysis of the positive monoclonal phage, 4 repeat amino acid sequences were obtained. The fluorescein isothiocyanate-labelled (FITC)-CSPLNTRFC with the highest repetition rate was synthesized for FITC-CSPLNTRFC peptide. Bcap-37 cells tumor-bearing nude mice model assay in vivo showed that FITC-CSPLNTRFC peptide could significantly enriched in the breast xenograft tissues. Conclusion CSPLNTRFC peptide specifically binding to human medullary breast cancer Bcap-37 cells can be screened out successfully by using phage display in vivo, which will be helpful to vitro studies on the early diagnosis of breast cancer. Key words: Breast neoplasms; Peptide library; Phage display; Specifically binding peptide

TRAIL receptors are differentially regulated and clinically significant in gallbladder cancer

Deregulation of the receptors of TNF-related apoptosis inducing ligand (TRAIL) has been reported in various cancers. In an effort to define the role of these receptors we profiled their expression in gallbladder cancer (GBC) and explored their clinical significance. Expression of TRAIL receptors' mRNA in GBC was analysed through reverse transcriptase polymerase chain reaction (RT-PCR), and protein through western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). mRNA data show frequent higher expression of TRAIL receptors in GBC samples. Death receptors DR4 and DR5 showed significant negative correlation with tumour stage, T stage and tumour grade; DcR1 transcript showed positive correlation with tumour stage, N stage, M stage and tumour grade. Similarly, IHC showed frequent positive staining for DR4, DR5 and DcR1in GBC samples. Cytoplasmic and nuclear DR4 protein showed negative correlation with T stage and tumour grade, whereas cytoplasmic DcR1 protein showed positive correlation with tumour stage and N stage. Nuclear DcR1 showed positive correlation with N stage. ELISA results showed significantly higher expression of secretory DcR1 in GBC patients. Kaplan-Meier analysis demonstrated significantly decreased mean survival of patients with positive staining of cytoplasmic DcR1. High level of death receptors identified the patients with early gallbladder cancer, whereas high DcR1 expression served as a prognostic factor for poor outcome.

Comparison of C3d immunohistochemical staining to enzyme-linked immunosorbent assay and immunofluorescence for diagnosis of bullous pemphigoid

Bullous pemphigoid (BP), the most common autoimmune blistering disease, may be diagnostically challenging. Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and recently, C3d immunohistochemistry (IHC), are used as adjuncts to diagnosis. To compare C3d IHC to DIF, IIF, and ELISA testing in BP diagnosis. C3d IHC was performed on skin biopsyspecimens from 51 patients (27 with BP and 24 with other blistering diseases) and compared to DIF and IIF, with anti-BP180 or anti-BP230 ELISA results used as the gold standard. We found C3d IHC, DIF, and IIF had similar sensitivity (74.1%, 63.1%, and 70.4%), specificity (95.8%, 100%, and 100%), positive predictive value (95.2%, 100%, and 100%), and negative predictive value (76.7%, 70.6%, and 75%) for BP. Cases with positive C3d IHC, DIF, and IIF had significantly higher anti-BP180 and anti-BP230 by ELISA than cases with negative testing (P<.0001). False-negative IIF results were associated with lower BP230 compared with true-positive results (P=.03). This was a single-center, retrospective study. Our study compared C3d IHC to DIF and IIF in BP diagnosis, demonstrating C3d IHC on fixed tissue provides similar diagnostic utility to immunofluorescence and ELISA.

Measuring serum human epididymis secretory protein autoantibody as an early biomarker of lung cancer.

BackgroundLung cancer (LC) is one of the most common types of malignant tumors and is the most prominent cause of tumor-related death worldwide. LC is a heterogeneous disease caused by somatic cell mutations and dysregulation in several signaling pathways. Understanding these pathways provides the basis for detecting LC. LC screening and diagnosis in current clinic still rely on computed tomography (CT), but its high false positive rates and cost may prevent it from being a routine screening method. Therefore, the discovery of new non-invasive and more valuable biomarkers may present an improved diagnostic approach for LC, and potentially provide more useful information for the prognosis and treatment of LC in patients. This study investigated the potential of detecting serum autoantibodies produced against human epididymis secretory protein 4 (HE4) for LC diagnosis in high-risk groups.MethodsSerum samples from 61 patients with LC were included in this study, and another 53 serum samples from healthy donors or benign lung diseases (BLD) patients were collected as the control group. The samples were analyzed with enzyme-linked immunosorbent assays (ELISA).ResultsELISA results showed significantly higher levels of serum autoantibodies against HE4 in samples from LC patients compared to the control group (P<0.001). Analysis of HE4 autoantibodies showed a receiver operating characteristic (ROC) curve indicating 67.21% sensitivity, 96.23% specificity, and an area under the curve (AUC) of 0.848. Levels of HE4 autoantibodies can discriminate early-stage LC patients from the control group with a 54.76% sensitivity.ConclusionsDetecting serum HE4 autoantibody levels may be a potential biomarker in high-risk groups of LC. We present a new method for the diagnosis of LC in the clinic.

MiR-137 affects bone mineral density in osteoporosis rats through regulating RUNX2.

To study the influence of micro ribonucleic acid (miR)-137 on osteoporosis rats by regulating runt-related transcription factor 2 (RUNX2). A total of 36 Sprague-Dawley rats were randomly assigned to the normal group (n=12), model group (n=12), and inhibitor group (n=12). No treatment was performed in the normal group. The osteoporosis model in rats was prepared in the model group, and miR-137 inhibitor was administered in osteoporosis rats of inhibitor group. Following 12 weeks of intervention, sampling was conducted. The expression of RUNX2 was detected via immunohistochemistry, and its protein expression level was determined via Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was carried out to detect the mRNA level of miR-137. The contents of serum bone Gla protein (BGP) and total alkaline phosphatase (TALP) were measured using enzyme-linked immunosorbent assay (ELISA). Finally, bone mineral density was determined with a dual-energy X-ray absorptiometry instrument. According to the immunohistochemistry detection, the rats in model group and inhibitor group had a notably lower positive expression level of RUNX2 than normal group (p<0.05), and its expression level in the inhibitor group was substantially higher than that in the model group (p<0.05). Western blotting results showed that compared with that in the normal group, the protein expression level of RUNX2 was notably lowered in the model and inhibitor group (p<0.05), which was markedly higher in the inhibitor group than that in the model group (p<0.05). It was found through the qPCR that the expression level of miR-137 was remarkably raised in both model group and inhibitor group compared with that in the normal group, showing statistically significant differences (p<0.05). The rats in the inhibitor group had a remarkably lower expression level of miR-137 than the model group (p<0.05). ELISA results revealed that the model group and inhibitor group had substantially lower contents of serum BGP and TALP than the normal group (p<0.05), and that their contents rose dramatically in the inhibitor group compared with that in the model group (p<0.05). Additionally, based on the measurement of bone mineral density, compared with that in the normal group, bone mineral density declined considerably in the model group and inhibitor group (p<0.05). It was markedly elevated in inhibitor group in comparison with that in the model group (p<0.05). MiR-137 regulates RUNX2 to affect the bone mineral density of osteoporosis model rats.

Investigating effect of Faeces Bombycis on intestinal microflora in rats with syndrome of damp retention in middle-jiao by high-throughput sequencing

The aim of this paper was to investigate the effect of Faeces Bombycis(FB) on the intestinal microflora in rats with syndrome of damp retention in middle-jiao, and to explore its mechanism in regulating intestinal microflora from the perspective of microorganisms contained in FB. The contents of antidiuretic hormone(ADH) and C-reactive protein(CRP) in serum and aquaporin 3(AQP3) in jejunum were determined by enzyme-linked immunosorbent assay(ELISA). Illumina Miseq platform was used for high-throughput sequencing of the rat feces and FB. The ELISA results showed that as compared with the normal control group, the contents of ADH and CRP in the model group were significantly increased(P&lt;0.05), and the content of AQP3 was significantly decreased(P&lt;0.05). After drug administration, the ADH, CRP and AQP3 contents were recovered. Sequencing of rat feces showed that the ACE, Chao1 and Shannon indexes of the intestinal microflora were the lowest in the model group. As compared with the normal control group, the levels from phylum to genus were all significantly changed in model group, and Proteobacteria, Acinetobacter, Anaerobacter, Pseudomonas, and Parabacteroides levels were significantly increased(P&lt;0.05), while Marvinbryantia level was significantly decreased(P&lt;0.05). As compared with the model group, Proteobacteria was significantly decreased in the FB low and high dose groups(P&lt;0.05), and Acinetobacter, Anaerobacter, Pseudomonas, Parabacteroides levels were significantly decreased in the low, medium and high dose groups(P&lt;0.05), while Lachnoanaerobaculum, Intestinimonas and Marvinbryantia were increased significantly in the high dose group(P&lt;0.05). Sequencing analysis of FB showed that the relative abundance of Leclercia, Pantoea, Brachybacterium, Shimwellia, Hartmannibacter, Klebsiella, Serratia, Aurantimonas, Paenibacillus and Bacillus was high in the FB, but they were basically not present or little in the rat feces. In conclusion, FB may play a role in the treatment of &quot;syndrome of damp retention in middle-jiao&quot; by balancing the intestinal microflora, and this effect may be related to the metabolites of microorganisms in the FB.