Effect of prostaglandins and their precursors on the proliferation of human lymphocytes and their secretion of tumor necrosis factor and various interleukins

Cytokines, released by T cells, participate in inflammation and produce tissue injury. Excess production of cytokines such as interleukins (ILs) and tumor necrosis factor (TNF) is believed to be involved in the pathobiology of conditions such as septicemia and septic shock, collagen vascular diseases, glomerulonephritis etc. On the other hand, prostaglandins (PGs) are known to modulate inflammation, immune response, and T-cell response to antigens. But relatively little information is available on the effects of PGs and PG precursors on the release of cytokines. Here the authors present data which suggests that PGs including thromboxane B₂ (TXB₂) and their precursors such as dihomo-gamma linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) can inhibit T-cell proliferation and influence their ability to secrete IL-2, IL-4, IL-6 and TNF in vitro. These results may have relevance to the use of PG-precursors in various inflammatory conditions including collagen vascular diseases.     

Blockade of tumor growth due to matrix metalloproteinase-9 inhibition is mediated by sequential activation of beta1-integrin, ERK, and NF-kappaB

We previously showed that matrix metalloproteinase (MMP)-9 inhibition using an adenovirus-mediated delivery of MMP-9 small interfering RNA (Ad-MMP-9), caused senescence in medulloblastoma cells. Regardless of whether or not Ad-MMP-9 would induce apoptosis, the possible signaling mechanism is still obscure. In this report, we demonstrate that Ad-MMP-9 induced apoptosis in DAOY cells as determined by propidium iodide and terminal deoxynucleotidyltransferase-mediated nick end labeling staining. Ad-MMP-9 infection induced the release of cytochrome c, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase. Ad-MMP-9 infection stimulated ERK, and electrophoretic mobility shift assay indicated an increase in NF-kappaB activation. ERK inhibition, using a kinase-dead mutant for ERK, ameliorated NF-kappaB activation and caspase-mediated apoptosis in Ad-MMP-9-infected cells. beta1-Integrin expression in Ad-MMP-9-infected cells also increased, and this increase was reversed by the reintroduction of MMP-9. We found that the addition of beta1 blocking antibodies inhibited Ad-MMP-9-induced ERK activation. Taken together, our results indicate that MMP-9 inhibition induces apoptosis due to altered beta1-integrin expression in medulloblastoma. In addition, ERK activation plays an active role in this process and functions upstream of NF-kappaB activation to initiate the apoptotic signal.     

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.     

Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.     

Precolumn o‐Phthalaldehyde‐N‐acetyl‐L‐cysteine Derivatization Followed by RP‐HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

An indirect reversed‐phase high‐performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP‐18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50–5000 ng/ mL for both enantiomers. The intra‐ and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments. Chirality 25:883–889, 2013. © 2013 Wiley Periodicals, Inc.     

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

Objectives

HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B27₂) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B27₂ can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B27₂–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.

Methods

The monoclonal HD5 antibody was selected from a phage library to target cell-surface B27₂ homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B27₂ and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.

Results

HD5 bound B27₂ with high specificity and affinity (K^d = 0.32 nM). HD5 blocked cell-surface interaction of B27₂ with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B27₂ interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B27₂ molecules.

Conclusion

HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B27₂ propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.     

Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ～14.3 μM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.