The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 µM NE by 50-60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 µM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 µM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.

Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18.

Interleukin (IL)-18 is an important effector of innate and adaptive immunity, but its expression must also be tightly regulated because it can potentiate lethal systemic inflammation and death. Healthy and septic human neonates demonstrate elevated serum concentrations of IL-18 compared with adults. Thus, we determined the contribution of IL-18 to lethality and its mechanism in a murine model of neonatal sepsis. We find that IL-18-null neonatal mice are highly protected from polymicrobial sepsis, whereas replenishing IL-18 increased lethality to sepsis or endotoxemia. Increased lethality depended on IL-1 receptor 1 (IL-1R1) signaling but not adaptive immunity. In genome-wide analyses of blood mRNA from septic human neonates, expression of the IL-17 receptor emerged as a critical regulatory node. Indeed, IL-18 administration in sepsis increased IL-17A production by murine intestinal γδT cells as well as Ly6G(+) myeloid cells, and blocking IL-17A reduced IL-18-potentiated mortality to both neonatal sepsis and endotoxemia. We conclude that IL-17A is a previously unrecognized effector of IL-18-mediated injury in neonatal sepsis and that disruption of the deleterious and tissue-destructive IL-18/IL-1/IL-17A axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.

Novel Role of Src in Priming Pyk2 Phosphorylation.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells.

The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.

Downregulation of c-SRC kinase CSK promotes castration resistant prostate cancer and pinpoints a novel disease subclass.

SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.

Reactive oxygen species in pathogenesis of atherosclerosis.

The volume of publications on the role of reactive oxygen species (ROS) in biological processes has been increasing exponentially over the last decades. ROS in large amounts clearly have detrimental effects on cell physiology, whereas low concentrations of ROS are permanently produced in cells and play a role as signaling molecules. An imbalance in ROS production and defense mechanisms can lead to pathological vascular remodeling, atherosclerosis being among them. The aim of this review is to examine different sources of ROS from the point of view of their participation in pathogenesis of atherosclerosis and related cardiovascular risk. Among the possible sources of ROS discussed here are mitochondria, NADPH-oxidases, xanthine oxidase, peroxidases, NO-synthases, cytochrome P450, cyclooxygenases, lipoxygenases, and hemoglobin of red blood cells. A great challenge for future research is to establish interrelations, feedback and feed-forward regulation mechanisms of various sources of ROS in development of atherosclerosis and other vascular pathologies.

Intradermal immunization improves protective efficacy of a novel TB vaccine candidate.

We have developed the Mycobacterium tuberculosis (Mtb) fusion protein (ID83), which contains the three Mtb proteins Rv1813, Rv3620 and Rv2608. We evaluated the immunogenicity and protective efficacy of ID83 in combination with several emulsion-formulated toll-like receptor agonists. The ID83 subunit vaccines containing synthetic TLR4 or TLR9 agonists generated a T helper-1 immune response and protected mice against challenge with Mtb regardless of route. The ID83 vaccine formulated with gardiquimod (a TLR7 agonist) also resulted in a protective response when administered intradermally, whereas the same vaccine given subcutaneously failed to provide protection. This highlights the need to explore different routes of immunization based on the adjuvant formulations used.

A comparative immunogenicity study of HIV-1 virus-like particles bearing various forms of envelope proteins, particles bearing no envelope and soluble monomeric gp120.

To assess the potential of native Envelope glycoprotein (Env) trimers as neutralizing antibody vaccines, we immunized guinea pigs with three types of VLPs and soluble gp120. Particles included "SOS-VLPs" (bearing disulfide-shackled functional trimers), "UNC-VLPs" (bearing uncleaved nonfunctional Env) and "naked VLPs" (bearing no Env). The SOS-VLPs were found to have a density of about 27 native trimers per particle, approximately twice that of live inactivated HIV-1 preparations. As immunogens, UNC- and SOS-VLP rapidly elicited anti-gp120 antibodies focused on the V3 loop and the gp120 coreceptor binding site. Reactivity to the gp41 immunodominant domain was absent in SOS-VLP sera, presumably because gp120-gp41 association is stabilized, effectively covering this epitope. Gp120-immune sera reacted with the receptor binding sites of gp120 and were less focused on the V3 loop. Some Env-VLP sera neutralized primary isolates at modest titers. The measurement of neutralization was found to be affected by the cell lines used. Depending on the assay particulars, non-Env specific antibodies in VLP sera could enhance infection, or nonspecifically neutralize. However, a neutralization assay using TZM-BL cells was essentially clear of these effects. We also describe a native trimer binding assay to confirm neutralization activity in a manner that completely eliminates nonspecific effects. Overall, our data suggests that Env-VLP sera were primarily focused on nonfunctional forms of Env on VLP surfaces, possibly gp120/gp41 monomers and not the trimers. Therefore, to make progress toward a more effective VLP-based vaccine, we will need to find ways to refocus the attention of B cells on native trimers.

Viral evolution in macaques coinfected with CCR5- and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies.

Macaques were immunized with SF162 Env-based gp140 immunogens and challenged simultaneously with the CCR5-tropic homologous SHIV(SF162P4) and the CXCR4-tropic heterologous SHIV(SF33A) viruses. Both mock-immunized and immunized animals became dually infected. Prior immunization preferentially reduced the viral replication of the homologous virus during primary infection but the relative replication of the two coinfecting viruses during chronic infection was unaffected by prior immunization, despite the fact that five of six immunized animals maintained a significantly lower overall viral replication that the control animals. Neutralizing antibodies participated in controlling the replication of SHIV(SF162P4), but not that of SHIV(SF33A). Dual infection resulted in the emergence and predominance within the circulating CCR5 virus pool, of a variant with a distinct neutralization phenotype. The signature of this variant was the presence of three amino acid changes in gp120, two of which were located in the receptor and coreceptor binding sites. Also, a significant fraction of the viruses circulating in the blood, as early as two weeks post-infection, was recombinants and prior immunization did not prevent their emergence. These findings provide new insights into the dynamic interaction of CCR5- and CXCR4-tropic HIV isolates that are potentially relevant in better understanding HIV-mediated pathogenesis.

The V1, V2, and V3 regions of the human immunodeficiency virus type 1 envelope differentially affect the viral phenotype in an isolate-dependent manner.

It is well documented that removal of the V1V2 region or of the V2 loop alone from the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) increases the susceptibility of these viruses to neutralization by antibodies. The specific role of the V1 loop in defining the neutralization susceptibility of HIV is, however, not well documented. Our current studies indicate that although the V1V2 region is a global modulator of the HIV-1 neutralization susceptibility, the individual roles the V1 and V2 loops have in defining the neutralization susceptibility profile of HIV-1 differ and in some cases are opposite. While deletion of the V2 loop renders the virus more susceptible to neutralization by antibodies that recognize diverse epitopes, in particular certain ones located in the CD4 binding site and the V3 loop, deletion of the V1 loop renders the virus refractory to neutralization, especially by antibodies that recognize CD4-induced epitopes and certain CD4-site binding antibodies. Our current studies also indicate that the relative involvement of the V2 loop of the HIV-1 envelope during virus-cell entry appears to be envelope background dependent. As a result, although deletion of the V2 loop from the clade B, R5-tropic SF162 HIV-1 virus resulted in a virus that was replication competent, the same modification introduced on the background of two other R5-tropic isolates, SF128A (clade B) or SF170 (clade A), abrogated the ability of these envelopes to mediate virus-cell entry.