Maurizio Costabile

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.

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. Hum Mutat 27(12), 1163–1173, 2006.

Measuring the 50% haemolytic complement (CH50) activity of serum.

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540 nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Recent advances in the development of sphingosine kinase inhibitors.

Sphingosine kinase (SK) 1 and 2 are lipid kinases that catalyse the formation of sphingosine 1-phosphate (S1P), a potent signalling molecule with a wide array of cellular effects. SK1 and 2 have been shown to be up-regulated in tumours and their genetic ablation or inhibition has been shown to slow tumour growth as well as sensitise cancer cells to chemotherapeutics. The SKs have been extensively studied, with a plethora of inhibitors developed that target the sphingosine-binding pocket of the enzyme, some with nanomolar affinities. Recently, inhibitors targeting the ATP pocket of SK have also been described. Here we discuss the development of these new small molecule SK inhibitors, summarise the recent discovery of off-targets effects of many current SK inhibitors, and provide an overview of the usefulness of these inhibitors as in vitro tools and therapeutic agents.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

IntroductionWhile 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.MethodsIn a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content.ResultsErythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content.ConclusionThe data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

Arachidonic acid and its COX1/2 metabolites inhibit interferon-γ mediated induction of indoleamine-2,3 dioxygenase in THP-1 cells and Human monocytes

Using human acute monocytic leukaemic THP-1 cells and human primary monocytes, this study examined the ability of arachidonic acid (AA) to modulate the activity of the IFNγ signalling cascade and its downstream effector indoleamine 2,3-dioxygenase (IDO). We established that AA inhibited IDO enzyme activity with an IC(50) of 20 μM in THP-1 cells and 12 μM in monocytes, and this was due to reduced expression of INDO1 mRNA and reduced level of IDO protein. Further mechanistic analysis revealed that AA interfered with the transcriptional function of the IFNγ signalling pathway by reducing phosphorylation of signal transducer and activator of transcription (STAT1) on tyrosine 701. The importance of AA metabolism via the COX and LOX pathways was investigated using inhibitors. Indomethacin, but not nordihydroguaiaretic acid, prevented the AA-mediated inhibition of STAT1 phosphorylation and thereby IDO enzymatic activity in THP-1 cells and monocytes. This is the first study to demonstrate that AA inhibits the IFNγ/STAT/IDO pathway, and this function is mediated by COX1/2 produced metabolites of AA. We now have evidence demonstrating that the AA metabolites, prostaglandins A(2) and D(2,) were highly inhibitory towards the IFNγ pathway, while prostaglandin E(2) had no effect. Together, these results indicate that the fatty acid AA has the potential to modulate the immunosuppressive activity of IDO and may form the basis of novel inhibitory compounds.

Inhibition of indoleamine 2,3-dioxygenase activity by fatty acids and prostaglandins: A structure function analysis.

Indoleamine 2,3-dioxygenase-1 (IDO-1) catalyses the first and rate-limiting step in the metabolism of L-tryptophan. Degradation of L-Trp leads to the production of several immunosuppressive metabolites, including N-formyl kynurenine and kynurenine (Kyn). Apart from a normal physiological role, IDO-1 has also been identified to play a crucial role in immune suppression and tumour induced tolerance. Indeed, many primary tumours express high levels of IDO-1 compared to normal cells of the same stroma. IDO-1 is accepted as being an inducible negative regulator of T cell viability, proliferation and activation. As such, IDO-1 has become a target of intense interest for pharmacological inhibition, for the treatment of cancer. We have previously demonstrated that AA and the prostaglandin metabolite, PGD2, repressed the IFNγ mediated activity of IDO-1 in THP-1 cells and human monocytes. In this study, we characterise the structure-function relationship of fatty acids and eicosanoids towards inhibition of IDO-1 activity in THP-1 cells and human monocytes. Using a commercial library of fatty acids, 55% of fatty acids inhibited IDO-1 activity. The activity of individual FAs was affected by chain length, number of double bonds and bond configuration. Interrogation of an AA derived eicosanoid library identified 13 PGs with significant inhibitory activity. A structure-function analysis revealed that the γ position of the cyclopentenone ring, double bond in the α-β position of the cyclopentenone ring, the presence of multiple OH groups in the side arm and the addition of an ethanolamide group, significantly increased the inhibitory activity of the PGs. Based on this data we have identified the structure of two possible compounds that may be even more potent pharmacological repressors of IDO-1.

Identification and functional characterization of two novel mutations in the α-helical loop (residues 484–503) of CYBB/gp91phox 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 p47phox and p67phox 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 gp91phox and the requirement of a negative charge at residue 500 in gp91phox 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.

Prostaglandin D2 is a novel repressor of IFNγ induced indoleamine-2,3-dioxygenase via the DP1 receptor and cAMP pathway.

Expression of elevated levels of Indoleamine 2,3-dioxygenase (IDO) is well established as a mechanism of cancer induced immunosuppression. Pharmacological inhibition of IDO activity is thus a promising alternative in the treatment of cancer. Previously we demonstrated that cyclooxygenase derived metabolites of arachidonic acid inhibited the interferon-gamma mediated induction of IDO in both THP-1 cells and human monocytes. Here we identified that of the five primary prostanoids produced by COX-1/COX-2, only PGD2 displayed significant repressor activity. PGD2 inhibited IDO activity with an IC50 of 7.2µM in THP-1 cells and 5.2µM in monocytes. PGD2 caused a significant decrease in both IDO mRNA and protein. Using receptor specific agonists, PGD2 was found to act via the DP1 receptor, while the CRTH2 receptor was not involved. A DP1 antagonist significantly reduced the activity of PGD2, while CRTH2 agonists were ineffective. PGD2 increased intracellular cAMP levels and exogenous N(6)-cAMP was also found to be highly inhibitory. The effects of PGD2 via cAMP were blocked by Rp-cAMP indicating involvement of PKA. PGD2 also stimulated CREB phosphorylation, a PKA dependent transcription factor. This is the first report demonstrating that PGD2, a prostanoid typically associated with allergy, can inhibit IDO activity via the DP1/cAMP/PKA/CREB pathway. Our findings suggest that PGD2 and its derivatives may form the basis of novel repressors of IFNγ-mediated IDO expression.

Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.