Use Of Immunofluorescence Assay Research Articles

Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation.

There are more than 70 million people worldwide living with epilepsy, with most experiencing the onset of epilepsy in childhood. Despite the availability of more than 20 anti-seizure medications, approximately 30% of epilepsy patients continue to experience unsatisfactory treatment outcomes. This situation places a heavy burden on patients' families and society. Childhood epilepsy is a significant chronic neurological disease that is closely related to genetics. Col4a2, the gene encoding the α2 chain of type IV collagen, is known to be associated with multiple diseases due to missense mutations. The Col4a2 variant of collagen type IV is associated with various phenotypes, including prenatal and neonatal intracranial hemorrhage, porencephaly, porencephaly with cataracts, focal cortical dysplasia, schizencephaly, strokes in childhood and adolescence, and sporadic delayed hemorrhagic stroke. Although epilepsy is recognized as a clinical manifestation of porencephaly, the specific mechanism of Col4a2-related epileptic phenotypes remains unclear. A total of 8 patients aged 2 years and 2 months to 18 years who were diagnosed with Col4a2-related infantile epileptic spasm syndrome were analyzed. The seizure onset age ranged from 3 to 10 months. Initial EEG results revealed hypsarrhythmia or multiple and multifocal sharp waves, spike waves, sharp slow waves, or spike slow waves. Elevated levels of the cytokines IL-1β (32.23±12.58 pg/ml) and IL-6 (45.12±16.03 pg/ml) were detected in the cerebrospinal fluid of these patients without any signs of infection. Following antiseizure treatment, decreased IL-1β and IL-6 levels in the cerebrospinal fluid were noted when seizures were under control. Furthermore, we aimed to investigate the role of Col4a2 mutations in the development of epilepsy. Through the use of immunofluorescence assays, ELISA, and Western blotting, we examined astrocyte activity and the expression of inflammatory cytokines such as IL-1β, IL-6, and TNF-α after overexpressing an unreported Col4a2 (c.1838G>T) mutant in CTX-TNA cells and primary astrocytes. We found that the levels of the inflammatory factors IL-1β, IL-6, and TNF-α were increased in both CTX-TNA cells (ELISA: p = 0.0087, p<0.001, p<0.001, respectively) and primary astrocytes (ELISA: p = 0.0275, p<0.001, p<0.001, respectively). Additionally, we conducted a preliminary investigation of the role of the JAK/STAT pathway in Col4a2 mutation-associated epilepsy. Col4a2 mutation stimulated astrocyte activation, increasing iNOS, COX-2, IL-1β, IL-6, and TNF-α levels in both CTX-TNA cells and primary astrocytes. This mutation also activated the JAK/STAT signaling pathway, leading to increased phosphorylation of JAK2 and STAT3. Treatment with the JAK/STAT inhibitor WP1066 effectively counteracted this effect in primary astrocytes and CTX-TNA cells. To date, the genes who mutations are known to cause developmental and epileptic encephalopathies (DEEs) are predominantly grouped into six subtypes according to function. Our study revealed that an unreported mutation site Col4a2Mut (c.1838G>T) of which can cause neuroinflammation, may be a type VII DEE-causing gene.

Immunofluorescence assay of pesticides on the base of immobilized multi-polyclonal antibody

The sensitive competitive immunofluorescence method for simultaneous determination of paraoxon and dichlorvos with immobilized multi-polyclonal antibody on magnetic nanoparticles was developed. The multi-polyclonal antibody was obtained after the immunization of mixture of two prepared immunogens dichlorvos-cBSA and paraoxon-BSA (1:1). The immunogens dichlorvos-cBSA and paraoxon-BSA were synthesized preliminary. Multi-polyclonal antibody against dichlorvos and paraoxon was covalently coupled on magnetic nanoparticles. The competitive fluorescence conjugates dichlorvos-cBSA-FITC and paraoxon-BSA-ATTO 620 were synthesized. Two typical calibration curves of immunofluorescence assay for determination of dichlorvos and paraoxon in buffer solutions were obtained. The linear interval from 2 to 200 ng/mL for these two pesticides was determined. Then the calibration curves for dichlorvos and paraoxon were obtained in cow milk solutions. The linear range of pesticides in cow milk was determined (from 5 to 300 ng/L) and the detection limit for paraoxon (3.5 ng/mL) and dichlorvos (4 ng/mL) was found. The obtained results for cow milk samples were compare with results in UHT, pasteurized cow milk, sheep and goal milk. Quite different are the results when analyzing paraoxon and dichlorvos in standard solutions prepared in sheep's milk. The linear working range for the two pesticides is between 7 and 300 ng/mL it is obviously, that there is a shift of all analytical characteristics up to higher values. The reason for this is the high fat content of sheep's milk. The obtained results were showed that the developed method was 2 time more sensitive than method with the results obtained with mixture (1:1) of two separated antibodies – anti-paraoxon and anti-dichlorvos. These results confirmed the potential of the immunoassay for quantitative simultaneous screening of both dichlorvos and paraoxon. Practical applications:The use of immunofluorescence assay based on immobilized multi-polyclonal antibody on magnetic nanoparticles allows us to detect simultaneous paraoxon and dichlorovos in raw milk and ather diary products.

Abstract A186: Novel target to control lung tumor growth: Disruption of cell membrane remodeling by modulating myoferlin expression.

Abstract Introduction: Lung cancer is the leading cause of cancer death in human. One feature of tumor cells is their ability to rapidly proliferate. These fast dividing tumor cells undergo continuous membrane damage and remodeling cycles. Hence, disruption of membrane remodeling and repair could block tumor growth. Myoferlin has been shown to mediate membrane processes such as receptor trafficking and membrane repair via endocytosis and exocytosis. Loss of myoferlin expression in non-cancer cell lines leads to defects in processes essential for cell proliferation, such as trafficking of membrane receptors (1, 2) and repairing ruptured cell membrane (3). Involvement of myoferlin in multifaceted membrane events shared by cancer pathogenesis suggests that modulating myoferlin expression can block tumorigenic activities. Herein, we investigated whether interfering with normal myoferlin expression, membrane repair and remodeling provides therapeutically relevant antitumor effects. Purpose and Hypothesis: Myoferlin regulates lung tumor growth by mediating cell proliferation and membrane remodeling process. Methods and Results: To investigate the potential expression of myoferlin in tumor, we performed Western blot analysis on a range of mouse and human cancer cell lines and immunohistochemistry on mouse and human lung carcinoma tissues. We found that myoferlin was expressed in various human and mouse cancer cell lines as well as solid tumors. With the use of immunofluorescent assay, we visualized the localization of myoferlin expression around the peri-nuclear region, cytoplasm and Golgi apparatus in cultured mouse Lewis lung carcinoma (LLC) cells. To assess the role of myoferlin in tumor pathogenesis, loss of function studies were performed using a myoferlin silencing RNA (siRNA)-based approach. Briefly, mouse Lewis lung carcinoma (LLC) cells were transfected with myoferlin siRNA and the effects of myoferlin knockdown on tumor cell proliferation and membrane repair were evaluated. A mouse xenograft tumor model was also used to determine the effects of myoferlin knockdown on solid tumor growth. Knockdown of myoferlin caused a 90% decrease in proliferation of mouse LLC cells and disabled membrane resealing after membrane damage. In addition, myoferlin siRNA decreased solid lung tumor growth by 55%, which was attributed to substantial reduction of tumor cell proliferation. Conclusion: Our results identify the anti-proliferative effect associated with attenuated myoferlin expression in lung tumor growth both in vitro and in vivo. This opens up a new therapeutic approach for lung cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A186. Citation Format: Suk Kei Cleo Leung, Carol Yu, Michelle I. Lin, Cristina Tognon, Pascal Bernatchez. Novel target to control lung tumor growth: Disruption of cell membrane remodeling by modulating myoferlin expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A186.

Transmission ofTrypanosoma cruziby Heart Transplantation

Trypanosoma cruzi infection (i.e., Chagas disease) is an unusual complication that can occur after solid-organ transplantation and that can result in severe illness or death. In 2006, there were 2 heart transplant recipients in Los Angeles, California, reported to have acute trypanosomiasis during the same month. We conducted an investigation to determine the source of these infections. We reviewed the medical, organ procurement, and donor transfusion and transplantation records of these 2 heart transplant recipients. The 2 heart transplant recipients were interviewed regarding any kind of natural exposure and were screened for parasites by obtaining blood and other tissue samples for buffy coat, culture, and polymerase chain reaction. Serum samples from the heart transplant recipients, organ donors, and blood donors were tested for T. cruzi antibodies by use of immunofluorescence assay and radioimmunoprecipitation assay. Tissue samples from the organ donors were examined by use of polymerase chain reaction and immunohistochemical staining. Other recipients of organs from the same donors were monitored for T. cruzi infection by use of polymerase chain reaction and immunofluorescence assay. Both heart transplant recipients had no apparent risk factors for preexisting T. cruzi infection. Both were seronegative but tested positive for the parasite, indicating recent infection. Both recipients died despite medical treatment. The organ donors tested positive for T. cruzi antibodies by use of radioimmunoprecipitation assay; the blood donors were seronegative. Six other patients had received a liver or kidney from these organ donors. None showed evidence of T. cruzi infection. To our knowledge, this is the first report of T. cruzi transmission associated with heart transplantation. Clinicians and public health authorities should be aware that manifestations of Chagas disease can occur after transplantation, requiring rapid evaluation, diagnosis, and treatment.

Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP.

Consumption of contaminated water has been implicated as a major source of Cryptosporidium infection in various outbreak investigations and case control studies. Surveys conducted in various regions of the United States demonstrated the presence of Cryptosporidium oocysts in 67-100% of wastewaters, 24-100% of surface waters, and 17-26.8% of drinking waters. The identity and human infective potential of these waterborne oocysts are not known, although it is likely that not all oocysts are from human-infecting Cryptosporidium species. Likewise, the source of the oocyst contamination is also not fully clear. Farm animals and human sewage discharge are generally considered to be the major sources of surface water contamination with C. parvum. Because Cryptosporidium infection is common in wildlife, it is conceivable that wildlife can also be a source for Cryptosporidium oocysts in waters. The presence of host-adapted Cryptosporidium spp. and genotypes makes it possible to develop molecular tools to assess the human infection potential and source of Cryptosporidium oocysts in water.Currently, the identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA) after concentration processes (Environmental Protection Agency [EPA] recommended information collection rule [ICR] method or method 1622/1623 or similar techniques). Because IFA detects oocysts from all Cryptosporidium parasites, the species distribution of Cryptosporidium parasites in environmental samples cannot be assessed. Although many surface water samples contain Cryptosporidium oocysts, it is unlikely that all these oocysts are from human-pathogenic species or genotypes, because only five genotypes of Cryptosporidium parasites (the C. parvum human and bovine genotypes, C. meleagridis, C. canis, and C. felis) are responsible for most human infections. Information on the source of C. parvum contamination is necessary for effective evaluation and selection of management practices for reducing C. parvum contamination of surface water and the risk of cryptosporidiosis. Thus, identification of oocysts to species and genotype levels is of public health importance.

Association between cancer chemotherapy and canine distemper virus, canine parvovirus, and rabies virus antibody titers in tumor-bearing dogs.

To determine the association between cancer chemotherapy and serum canine distemper virus (CDV), canine parvovirus (CPV), and rabies virus antibody titers in tumor-bearing dogs. Prospective study. 21 client-owned dogs with various malignancies and 16 client-owned dogs with lymphoma. In study A, serum antibody titers were measured by use of hemagglutination inhibition (CPV titers) or serum neutralization (CDV titers) before and at least 1 month after initiation of chemotherapy. Baseline values were compared with values obtained from a control population of 122 healthy dogs seen for routine revaccination. Titers were considered protective at > or = 1:96 for CDV and > or = 1:80 for CPV. In study B, serum IgG titers were measured by use of immunofluorescent assay (CDV and CPV titers) and rapid fluorescent focus inhibition test (RFFIT, rabies titers) at baseline and again at weeks 5, 8, and 24 of a standard chemotherapy protocol for treatment of lymphoma. An IgG titer of > or = 1:50 was considered protective for CPV and CDV. An RFFIT titer of > or = 0.5 U/ml was considered protective for rabies virus. Significant changes were not detected in CDV, CPV, and rabies virus titers following chemotherapy in tumor-bearing dogs. Results suggest that established immunity to CDV, CPV, and rabies virus from previous vaccination is not significantly compromised by standard chemotherapy used to treat tumor-bearing dogs.

An evaluation of the tissue expression of a bovine dodecapeptide bactenecin in the adult and fetal animal

Various antimicrobial cationic peptides have been isolated from the granules of bovine neutrophils. One of these peptides is only 12 amino acids in length and is called a dodecapeptide bactenecin. A monoclonal antibody to the synthetic bovine dodecapeptide bactenecin was produced, characterised and used in an immunofluorescent assay to assess the peptide's expression. When adult bovine peripheral blood and both adult and fetal bone marrow, tracheal lining, liver, spleen and intestinal tissues were examined, the peptide was found only within neutrophils and cells within the bone marrow. These findings both confirm and supplement another report in the literature (Storici et al. 1992), however this is the first report of a bovine antimicrobial peptide detected within the bone marrow of fetal mammals. Our results also suggest the use of immunofluorescence assays to be especially well suited for the quick screening of antimicrobial peptide tissue expression.

Prevalence of Serum Antibodies to Hantaviruses in Northern Sweden as Measured by Recombinant Nucleocapsid Proteins

An enzyme-linked immunosorbent assay (ELISA) based on recombinant nucleocapsid protein (rN delta) (aa 1-117) of Hantaan, Seoul, Dobrava, Sin Nombre and Puumala hantaviruses was used to determine the prevalence of antibodies among randomized and stratified individuals from northern Sweden. In total, 137/1533 individuals (8.9%) had specific serum IgG antibodies to Puumala virus, the only hantavirus known to occur in the region. The prevalence of antibodies to Puumala virus (8.9%) was determined to be higher than previously reported (5.4%) in the same serum material, by use of immunofluorescence assay. As expected, sera reactive to Puumala virus rN delta did frequently cross-react with Sin Nombre virus protein. Unexpectedly, 21/1533 (1.4%) individuals recognized the Sin Nombre virus rN delta exclusively. Another 8 subjects showed reactivity in the ELISA to Hantaan, Seoul, or Dobrava virus-derived rN delta but not Puumala virus or Sin Nombre virus rN delta. The present demonstration in some individuals of antibodies specifically recognizing the Sin Nombre, Dobrava, Hantaan and Seoul virus protein justifies an awareness of the possibility that hantaviruses antigenically different from Puumala virus might occur in the region.

Seroimmunology of aids retrovirus infection I. Use of immunofluorescence assay to confirm sera with elisa reactivity

One thousand sera shown to be reactive by one of two commercial enzyme linked immunosorbent assays (ELISA) for antibodies to the AIDS virus were referred to the NSW State Reference Laboratory for confirmatory assays. Each serum was retested by two commercial ELISA systems (Abbott and ENI), the ENI exclusionary H9 ELISA and an immunofluorescence assay. Three hundred and twenty four sera were reactive by all 3 tests whereas 244 demonstrated concordant non-reactivity. Three hundred and seventy seven sera were reactive by Abbott EIA only and could not be confirmed positive by the ENI ELISA incorporating exclusionary testing, immunofluorescence or Western immunoblot of representative sera. Sera obtained from teaching hospital laboratories were more likely to be positive and less likely to be negative by all 3 tests, and were also less likely to be Abbott EIA reactive only compared with sera obtained from the blood transfusion service. Of the remaining 55 sera, 52 demonstrated a negative immunofluorescent reaction or a pattern of equal fluorescence on AIDS virus infected and control cells. Representative sera were shown to be negative on Western immunoblot analysis. Of the 3 sera which demonstrated immunofluorescence reactivity, one was positive and one was negative by Western immunoblot, and one could not be determined. We conclude that a combination of two ELISAs, one with an exclusionary ELISA test and an immunofluorescence assay is a reliable and simple means of confirming reactive sera for AIDS virus antibodies.

The Presence of Anti-Glomerular Basement Membrane Antibodies in Peripheral Blood

Abstract The use of immunofluorescent assay for circulating anti-glomerular basement membrane (anti GBM) antibodies is described. Groups of people studied for the presence of anti-GBM antibody included: a) normals; b) immune-complex glomerulonephritic patients; c) anti-GBM antibody-mediated nephritic patients; d) a hemodialysis-maintained group of kidney disease patients; and e) an allergy clinic group. Anti-GBM antibodies were found in the circulation only in patients with anti-GBM antibody-mediated nephritis. Paired-label isotopic studies to determine the percentage fixation of kidney-fixing antibodies, combined with serial dilution of the same anti-GBM antibody-containing sera, allowed estimation of the sensitivity of the assay to be in the range of 0.1 to 1.0 µg/ml antibody content. Despite the sensitivity of the technique and its specificity, two principal handicaps were encountered: a) only 40% of people known to have anti-GBM antibody-mediated glomerulonephritis were shown to have circulating anti-GBM antibodies, and b) several sera were encountered which fixed with wide anatomical definition to the target substrate and made it difficult to recognize the presence of anti-GBM antibodies in such sera.