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In JoVE (2)
- Determining the Reactivity and Titre of Serum using a Haemagglutination Assay
- Measuring the 50% Haemolytic Complement (CH50) Activity of Serum
Other Publications (9)
Articles by Maurizio Costabile in JoVE
JoVE Immunology and Infection
Determining the Reactivity and Titre of Serum using a Haemagglutination Assay
School of Pharmacy and Medical Sciences, University of South Australia
Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.
JoVE Immunology and Infection
Measuring the 50% Haemolytic Complement (CH50) Activity of Serum
School of Pharmacy and Medical Sciences, University of South Australia
The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.
Other articles by Maurizio Costabile on PubMed
Selective Deficiency in Protein Kinase C Isoenzyme Expression and Inadequacy in Mitogen-activated Protein Kinase Activation in Cord Blood T Cells
The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKC beta I, epsilon, theta and zeta. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4 h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.
Unique Effect of Arachidonic Acid on Human Neutrophil TNF Receptor Expression: Up-regulation Involving Protein Kinase C, Extracellular Signal-regulated Kinase, and Phospholipase A2
Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.
Characterization of the MEK5-ERK5 Module in Human Neutrophils and Its Relationship to ERK1/ERK2 in the Chemotactic Response
The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3-5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.
The Immunomodulatory Effects of Novel Beta-oxa, Beta-thia, and Gamma-thia Polyunsaturated Fatty Acids on Human T Lymphocyte Proliferation, Cytokine Production, and Activation of Protein Kinase C and MAPKs
We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (beta-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of beta-oxa, beta-thia, and gamma-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated beta-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-beta production. However, beta-oxa 25:6n-3 and beta-oxa 21:4n-3 displayed lower inhibitory action on IFN-gamma production. Surprisingly, beta-oxa 23:4n-6 and beta-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The beta-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two gamma-thia PUFA tested, gamma-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but gamma-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, beta-thia 23:4n-6 and beta-thia 21:3n-6, were studied in more detail and shown to have an IC(50) of 1-2 muM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.
Molecular Approaches in the Diagnosis of Primary Immunodeficiency Diseases
Over 120 inherited primary immunodeficiency diseases (PIDs) are known to exist. The genes responsible for many of these diseases have also been identified. Recent advances in diagnostic procedures have enabled these to be identified earlier and appropriately treated. While a number of approaches are available to identify mutations, direct sequencing remains the gold standard. This approach identifies the exact genetic change with substantial precision. We suggest that a sensitive and economical approach to mutation detection could be the direct sequencing of cDNA followed by the confirmatory sequencing of the corresponding exon. While screening techniques such as single-stranded conformation polymorphism (SSCP), heteroduplex analysis (HA), denaturing gradient gel electrophoresis (DGGE), and denaturing high-performance liquid chromatography (dHPLC) have proven useful, each has inherent advantages and disadvantages. We discuss these advantages and disadvantages and also discuss the potential of future sequencing technologies such as pyrosequencing, combinatorial sequencing-by-hybridization, multiplex polymerase colony (polony), and resequencing arrays as tools for future mutation detection. In addition we briefly discuss several high-throughput SNP detection technologies.
Capture and Generation of Adenovirus Specific T Cells for Adoptive Immunotherapy
Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti-viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon-gamma (IFN-gamma) secretion and capture assay to isolate ADV-specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus-specific T cells for cytomegalovirus or Epstein-Barr virus, the ADV response was dominated by CD4(+) T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN-gamma expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV-pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross-reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV-specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.
Novel Action of N-3 Polyunsaturated Fatty Acids: Inhibition of Arachidonic Acid-induced Increase in Tumor Necrosis Factor Receptor Expression on Neutrophils and a Role for Proteases
Neutrophils and tumor necrosis factor (TNF) play important roles in the pathogenesis of rheumatoid arthritis (RA). Modulation of TNF receptors (TNFRs) may contribute to the regulation of tissue damage, and n-6 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) can increase the expression of TNFRI and TNFRII on neutrophils. Because the n-3 PUFAs are antiinflammatory in RA, we examined whether, as a novel mechanism of action, n-3 PUFAs can antagonize the AA-induced increase in TNFR expression.
Leukocyte Numbers and Function in Subjects Eating N-3 Enriched Foods: Selective Depression of Natural Killer Cell Levels
While consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation.
Identification and Functional Characterization of Two Novel Mutations in the α-helical Loop (residues 484-503) of CYBB/gp91(phox) Resulting in the Rare X91(+) Variant of Chronic Granulomatous Disease
Chronic granulomatous disease (CGD) is mainly caused by mutations in X-linked CYBB that encodes gp91. We have identified two novel mutations in CYBB resulting in the rare X91(+) -CGD variant, c.1500T>G (p.Asp500Glu) in two male siblings and c.1463C>A (p.Ala488Asp) in an unrelated male. Zymosan and/or PMA (Phorbol 12-myristate 13-acetate)-induced recruitment of p47(phox) and p67(phox) to the membrane fraction was normal for both mutants. Cell-free assays using recombinant wild-type and the mutant proteins revealed that these mutants were not activated by NADPH (nicotinamide adenine dinucleotide phosphate). Interestingly, the Ala488Asp mutant was activated by NADPH in the presence of glutathione. These data suggest that the mutations prevented NADPH from binding to gp91(phox) and the requirement of a negative charge at residue 500 in gp91(phox) for NADPH oxidase assembly, in contrast to a previously described Asp500Gly change. These mutations and the effect of glutathione provide a unique insight into disease pathogenesis and potential therapy in variant X91(+) -CGD. Hum Mutat 33:471-475, 2012. © 2011 Wiley Periodicals, Inc.
