Enzyme-linked Immnosorbent Assay Research Articles

Inhibition of Phosphodiesterase 4 by FCPR03 Alleviates Chronic Unpredictable Mild Stress-Induced Depressive-Like Behaviors and Prevents Dendritic Spine Loss in Mice Hippocampi

BackgroundPhosphodiesterase 4 is a promising target for developing novel antidepressants. However, prototype phosphodiesterase 4 inhibitors show severe side effects, including nausea and vomiting. N-Isopropyl-3-(cyclopropylmethoxy)-4-difluoromethoxy benzamide (FCPR03) is a novel phosphodiesterase 4 inhibitor with little emetic potential. In the present study, we investigated the inhibitory effect of FCPR03 on chronic unpredictable mild stress-induced, depressive-like behaviors in mice and explored the underlying mechanisms.MethodsThe depression model of mice was established by chronic unpredictable mild stress. Forced swim test, tail suspension test, and sucrose preference test were used to assess depressive-like behaviors. Golgi-staining was utilized to analyze dendritic morphology and spine density. The level of cAMP was measured by enzyme-linked immnosorbent assay assay. Western blot was used to evaluate protein levels of phosphorylated cAMP-response element binding protein, protein kinase B, glycogen synthase kinase-3β, and brain derived neurotrophic factor in both hippocampus and prefrontal cortex. Postsynaptic density protein 95 and synapsin 1 were also detected by western blot in the hippocampi.ResultsTreatment with FCPR03 (0.5–1.0 mg/kg, i.p.) increased consumption of sucrose in the sucrose preference test in mice exposed to chronic unpredictable mild stress. FCPR03 shortened the immobility time in forced swim test and tail suspension test without affecting locomotor activity. Furthermore, chronic unpredictable mild stress decreased the dendritic spine density and dendritic length in the hippocampus. This change was accompanied by decreased expression of postsynaptic density protein 95 and synapsin 1. Interestingly, FCPR03 prevented dendritic spine loss and increased synaptic protein levels. Moreover, the levels of cAMP, phosphorylated cAMP-response element binding protein, and brain derived neurotrophic factor were elevated in chronic unpredictable mild stress-challenged mice after treatment with FCPR03. In addition, FCPR03 also enhanced the phosphorylation of both protein kinase B and glycogen synthase kinase-3β in mice exposed to chronic unpredictable mild stress.ConclusionThe present study suggests that FCPR03 could prevent both depressive-like behaviors and spine loss induced by chronic unpredictable mild stress in the mice hippocampi.

A Comprehensive in Silico Analysis of Regulatory SNPs of Human CLEC7A Gene and Its Validation as Genotypic and Phenotypic Disease Marker in Recurrent Vulvovaginal Infections.

Recurrent Vulvovaginal infections (RVVI) are the commonly reported microbiological syndrome affecting millions of women globally. Various molecules of innate immune system are instrumental in clearance of these microbial pathogens, thus suggested as one of the most important contributing factor in determining the disease outcome. Dendritic cell-associated C-type lectin-1 (Dectin-1) is an important molecule of innate immunity that is primarily known for its role in antifungal defenses. However, role of dectin-1 in recognition of other pathogens is also documented. The intracellular expression of dectin-1 was shown to be up-regulated by Mannose Binding Lectin (MBL)-mediated opsonophagocytosis of pathogens. Dectin-1 is encoded by CLEC7A, postulated to be a candidate gene in modulating risk of developing RVVI. In this study, we identified CLEC7A causal variants using in silico analysis. To assess their impact on susceptibility to RVVI, these causal variants along with serum dectin-1 levels (sDectin-1) were investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and Enzyme Linked Immnosorbent Assay (ELISA) respectively, under a case-control design. Furthermore, effect of these polymorphisms was also assessed on sMBL levels. In silico analysis revealed 9 putative functional conserved SNPs of CLEC7A. Association analysis revealed a significantly lower risk of developing RVVI and its types in carriers of CLEC7A rs3901533 G allele and its homozygous genotypes (p < 0.05). The heterozygous genotype was associated with significant protection against RVVI (p = 0.004). Haplotypes GGG and GTA showed significant protection against RVVI (p < 0.0001; p = 0.0003), Bacterial Vaginosis (p = 0.03; p = 0.002), Vulvovaginal Candidiasis (p = 0.03; p = 0.01) and Mixed Infections (p = 0.007; p = 0.04). Mean sDectin-1 levels were significantly high in RVVI and its types compared to controls (p < 0.05). Further, genotype-phenotype stratification showed significant differences within/between cases groups and controls. The CLEC7A rs3901533 polymorphism was also found to be associated with sMBL levels. The present study contributed novel insights into the role of dectin-1 in RVVI. CLEC7A rs3901533 polymorphism and high sDectin-1 levels along with low sMBL levels were found to be associated with RVVI susceptibility. Thus, screening of women with RVVI for these novel associations may lead to better diagnosis and treatment. Also genotyping method used in this study constitutes a simple and reliable assay, which can be confidently, used as a cheaper alternative for genotyping these variants in clinical settings. Finally, new restorative markers for other infectious diseases might be found by exploring nine functionally identified CLEC7A SNPs.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of &amp;lt;i&amp;gt;Prunus yedoensis&amp;lt;/i&amp;gt; Using Protoplast Co-Culture Bioassay Method

Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated. Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.

Genetic and Phenotypic Screening of Mannose-Binding Lectin in Relation to Risk of Recurrent Vulvovaginal Infections in Women of North India: A Prospective Cohort Study.

Recurrent Vulvovaginal Infections (RVVI) is common problem associated with women of reproductive age. The function and deleterious effect of Mannose Binding Lectin 2 (MBL2) common polymorphisms are reported to be associated with various diseases. However, the role of MBL2 promoter gene polymorphisms and their combined effect with structural variant along with Serum Mannose Binding Lectin (sMBL) levels in RVVI has not been investigated. The study included 258 RVVI cases and 203 age matched healthy controls. These were investigated for the distribution of MBL2 codon 54 and promoter polymorphisms by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). sMBL levels were quantified by Enzyme Linked Immnosorbent Assay (ELISA). The frequency of X allele and its genotypes was significantly high in cases than controls conferring risk toward RVVI and its types (p < 0.05). The HXPA (OR; 2.0), LXQB (OR; 1.43) haplotypes were associated with susceptibility to RVVI cases while haplotype LYQB significantly protected against RVVI (OR; 0.58), Bacterial Vaginosis (BV) (OR; 0.27) and Mixed Infections (MI) cases (OR; 0.62) with high frequency observed in controls (p < 0.05). Mean sMBL levels were significantly low in RVVI, BV, Vulvovaginal Candidiasis (VVC), and MI cases compared to controls (p < 0.05). VVC patient showed significantly low sMBL levels than RVVI and MI cases (p < 0.05). The mean sMBL levels segregated based on MBL2 genotypes and haplotypes showed significant difference in different cases groups with controls. The findings of the present study suggested that MBL2 Y/X polymorphism and low sMBL levels were associated with susceptibility to RVVI either it is BV, VVC, or MI. Thus MBL deficiency in women with RVVI may contribute to decreased efficiency in clearing of pathogens. Hence, specific measures like administration of purified or recombinant MBL might decrease the incidence of vaginal infections recurrences and more-effective treatment.

West Nile Virus Survey of Birds, Horses, and Mosquitoes of the Pacific Coast, Southern Mexico

Serology of West Nile virus vectors and non-human reservoirs was surveyed at Acapulco, Jose Azueta, and Ometepec, three Pacific Coast localities of Guerrero State, Mexico. The objectives of this study were to use enzyme-linked immnosorbent assay (ELISA) to assess West Nile virus antibodies of bird and equine serum samples and use reverse transcription of polymerase chain reaction (RT-PCR) to detect the virus in field-collected resting mosquitoes. Forty birds trapped using mist nets yielded 10% seroprevalence. Similarly, 18.6% of 102 equine blood samples had West Nile virus. In addition, 4,854 mosquitoes were caught using motorized backpack aspirators and grouped into 116 pools. Of the 16 species and seven genera, no mosquito was positive for West Nile virus. Our study demonstrated West Nile virus seroprevalence on resident birds and equines in Guerrero State, Mexico.

Effects of hypertonic saline on macrophage migration inhibitory factor in traumatic conditions

Trauma-induced suppression of cellular immune function contributes to sepsis, multiple organ dysfunction syndrome (MODS) and mortality. Macrophage migration inhibitory factor (MIF) has been revealed to be central to several immune responses. However, the role of MIF in trauma-like conditions is unknown. Therefore, the present study evaluated MIF in macrophages and polymorphonuclear neutrophils (PMNs). The effects of hypertonic saline (HTS) on lipopolysaccharide (LPS)-induced MIF levels were evaluated in macrophages. MIF concentrations were determined by an enzyme-linked immnosorbent assay (ELISA) and cell lysates were used for western blot analysis. The effects of HTS on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced MIF were evaluated in PMNs. MIF concentrations were determined by ELISA, western blotting and real time-polymerase chain reaction (RT-PCR) to determine MIF expression. MIF levels, which were measured by the ELISA, increased by 1.24±0.38 ng/ml in the supernatants of LPS-stimulated macrophages compared with the controls (0.79±0.07 ng/ml) at 2 h. HTS10 (150 mmol/l) partially restored MIF levels (0.84±0.22 ng/ml; P<0.05). Also, western blotting was performed and MIF protein levels were higher in the LPS-stimulated macrphages (20% increase in band density) compared with the controls (P<0.05). The addition of HTS decreased MIF protein expression. MIF levels in fMLP-stimulated PMN cells were unchanged compared with the controls according to the ELISA, western blotting and RT-PCR. No effects were observed following treatment with HTS. MIF concentrations and MIF expression were higher in LPS-stimulated macrophages than controls and HTS restored MIF levels to those of the controls. MIF levels were unchanged in PMNs stimulated by fMLP.

Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay

BackgroundProgress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic.MethodsGlutathione S-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation.ResultsThe results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG.ConclusionsThis study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

Molecular identities of human sperm proteins reactive with antibodies in sera of immunoinfertile women

Antisperm antibodies (ASA) can cause infertility in both men and women. It is important to delineate the sperm antigens against which these ASA are directed. Sperm proteins were separated by 2D gel electrophoresis and transferred to nitrocellulose membrane and incubated with sera from fertile women or immunoinfertile women having ASA. The corresponding immunoreactive peptide spots were cored from the gel and analyzed by the two-dimensional (2D) gel electrophoresis/matrix-assisted laser desoprtion ionization-time of flight-mass spectrometry and liquid chromatography-mass spectrometry (MALDI-TOF-MS/LC-MS). A total of 68 spots belonging to 38 different proteins and their isomers were identified. Fourteen of these proteins and their isomers reacted with both the fertile and immunoinfertile sera. Twenty-four of these proteins reacted specifically only with the immunoinfertile sera and not with the fertile sera. Among them was a novel protein designated as a hypothetical protein FLJ32704 (accession # Q96MA6). An immunodominant sequence (amino acid 151-159) of this protein was identified and a nonamer peptide based upon this sequence (IQTLG1TPR) was synthesized and examined for its immunoreactivity. This synthetic peptide reacted with 90% (36/40) of immunoinfertile sera and not with any of the fertile sera (0/40) in the enzyme-linked immnosorbent assay (ELISA). In conclusion, using the 2D gel electrophoresis/MALDI-TOF-MS/LC-MS procedure, we have identified several known and at least one novel antigen against which the antibodies are present in sera of immunoinfertile but not fertile women. Some of these antigens may find applications in specific diagonsis and treatment of infertility/immunoinfertility, and in the development of new generation of contraceptive modalities including contraceptive vaccines.

Detection of IgG Antibodies to Cryptosporidium parvum Using Eimeria tenella and Cryptosporidium parvum Antigens

Rahbari, S., Nabian, S., Azizi, H.R., Rezazadeh, A. and Mokhber-Dezfouli, M.R. 2005. Detection of IgG antibodies to Cryptosporidium parvum using Eimeria tenella and Cryptosporidium parvum antigens. J. Appl. Anim. Res., 28: 141–143. This paper describes and compares the use of Cryptosporidium parvum and Eimeria tenella oocyst antigens in application of enzyme linked immnosorbent assay (ELISA). The optical density (OD) mean value for positive Cryptosporidium sera with two antigens of Eimeria and Cryptosporidium were 0.476 and 0.310, respectively and sensitivity of both antigens was 100%. Cryptosporidium infection was detected in 79 (57%) cattle out of 137 fecal samples examined microscopically. The seroprevalence of IgG antibodies to Cryptosporidium parvum by using antigens of either Cryptosporidium or Eimeria were correlated with oocysts excretion. It is concluded that Eimeria antigen may be used for rapid screening of Cryptosporidium in calf diarrhea.

Effects of Fluorequinolone Treatment Acidified Feed, and Improved Hygiene Measures on the Occurrence of Salmonella Typhimurium DT104 in an Integrated Pig Breeding Herd

Worldwide, the use of antimicrobials in food production has been associated with drug resistance in foodborne pathogens such as Salmonella. However, little is known about the efficaciousness of fluorequinolone treatment on Salmonella Typhimurium T104 infections in pig breeding herds. A combined eradication procedure with enrofloxacin application on sows and piglets, feeding of encapsulated organic acids to sows, disinfection with peracetic acid, separation of the growers from the sows and serological discrimination using a new whole-cell-based enzyme-linked immnosorbent assay (ELISA) was evaluated for the suitability to eradicate and to control endemic S. Typhimurium DT104 infections in a closed herd. Thirty-seven sows and their piglets were treated everyday from day 14 ante partum until the day of weaning. Eighteen sows and their piglets served as controls. From the first day of life until day 168 after birth, faecal samples (n = 1671) of all piglets were analysed for Salmonella shedding. In parallel, systemic antibody responses were monitored by whole-cell-based isotype-specific ELISA systems. From birth to weaning the prevalence in both groups was between 2% and 9%. After weaning, intermittent shedding could be observed in both groups, and salmonellae could be found in up to 7.7% of the faecal samples. As a result, a dramatic increase in Salmonella-infected growers was observed, as of day 115 after birth, 47.4% of the animals of the treated group were tested positive for S. Typhimurium. Our results indicate that despite long-term antibiosis treatment and optimized hygiene measures, shedding of S. Typhimurium by the sows and the subsequent infection of their offspring could not be effectively prevented. Although it could be not shown that elimination of S. Typhimurium DT104 infection was achieved, the disinfection procedures described and the diagnostic test used are effective instruments to decrease the Salmonella load and to identify individual infected animals. Both of these are important factors for an improved consumer protection.

ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS BY IMMUNE COMPLEX: POSSIBLE INVOLVEMENT IN THE DEVELOPMENT OF TRANSFUSION-RELATED ACUTE LUNG INJURY

Background: Human polymorphonuclear neutrophils (PMNs) are suspected as a causative factor in the development of febrile nonhemolytic transfusion reactions (FNHTRs), such as transfusion-related acute lung injury (TRALI), although the mechanisms underlying FNHTRs have not yet been precisely clarified. The aim of this study was to evaluate the potency of immune complexes (ICs) to activate PMNs and to identify soluble factors produced by PMNs. Methods We: investigated the production of tumor necrosis factor α (TNFα) and perform by PMNs on stimulation with artificial oligomerized ICs by enzymelinked immnosorbent assay (ELISA). Furthermore, the potency of culture supernatants of PMNs that were stimulated with ICs was investigated with regard to the growth inhibitory activity against lung-tissue-derived primary cell culture (LT cells) and cytotoxic activity against LT cells by 3H-thymidine uptake experiment and 4-hour 51Cr release assay, respectively. Findings: PMNs activated by ICs were proved to produce TNFα and perform. The growth of LT cells was inhibited in the presence of cell-free culture supernatants of PMNs cultivated with ICs. Moreover, cell-free culture supernatants of PMNs cultivated with ICs appeared to have cytotoxic activity against LT cells. Conclusion: These results suggest that IC-stimulated PMNs produce inflammatory and cytotoxic mediators, which are potent inducers of FNHTRs such as TRALI.

Association between levels of TNF-alpha and TNF-alpha promoter -308 A/A polymorphism in children with Kawasaki disease.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to play a central role in the pathogenesis of vasculitis in Kawasaki disease (KD). We investigated the serum levels of TNF-alpha and soluble TNF receptor 1 (STNFR1) levels, and genetic polymorphisms of the TNF-alpha promoter gene in children with KD to delineate the genetic basis of KD. A total of 18 children (12 boys and 6 girls) with KD were studied, 9 of whom had the complication of coronary artery lesion (CAL) within 30 days after the onset of symptoms. Serum levels of TNF-alpha and STNFR1 were assayed by enzyme-linked immnosorbent assay, and DNA polymorphisms of the 5' flanking region of TNF-alpha promoter gene at position -308 [guanine (G) to adenine (A)] and -238 (G to A) were studied by direct nucleotide sequencing. The serum TNF-alpha level in KD patients was 113 +/- 209.9 pg/mL (range, 2.0 to 756.9 pg/mL; median, 24.7 pg/mL; normal, < 10 pg/mL). The serum levels of STNFR1 in KD (4255 +/- 2425 pg/mL) were higher than those of the control group (160 +/- 116 pg/mL). Allele frequencies of -308A and -238A were 11.1% and 0% in the KD patients, and 0% and 3.1% in the control group. Neither TNF-alpha promoter polymorphism nor any significant risk factor for CAL was identified in KD patients. One patient, who was homozygous for -308A, showed the highest TNF-alpha level and elevated STNFR1 level but had no evidence of CAL. Positive correlations were found between serum levels of STNFR1 and C-reactive protein (r = 0.731, p = 0.007), and between STNFR1 and leukocyte counts at admission (r = 0.620, p = 0.008). Increased serum levels of TNF-alpha and STNFR1 were found in KD patients but there was no correlation between these levels. The relationship between the pathogenesis of KD and TNF-alpha gene promoter -308G to A mutation towards cytokine production remains to be clarified.

Polymerase chain reaction-based diagnosis of mollicture infection of commercial animal sera

A total of 72 commercial animal sera were assayed for mollicute infection by a polymerase chain reaction (PCR)-based detection method and by four classical detection methods. The methods included microbiological assay by inoculation onto agar and into broth, DNA staining of an indicator cell line with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) fluorochrome, enzyme-linked immnosorbent assay (ELISA) and adenosine phosphorylase (AdoP) screening. Mollicutes were detected in 2 of the 72 sera assayed. The species isolated was Acholeplasma laidlawii in both cases. When detection methods were compared, PCR, microbiological culture and DNA staining were perfectly concordant. ELISA and AdoP detection produced false-negative results for 1 of the 2 infected sera each. False positive results appeared in 1 of the 70 mollicute-free sera with ELISA and in 16 of the 70 with AdoP detection. It was concluded that the PCR-based detection method is a useful tool in addition to current methods of detection of mollicutes in animal sera, with regard to reliability, sensitivity, specificity and time.