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Biological psychiatry  2012;73(7):613-621.
According to clinical studies, depression and cerebrovascular disease influence each other. Despite this evidence, no studies have investigated the relationship between major depressive disorder (MDD) and cerebrovascular disease at the cellular level. Astrocytic processes are a crucial interface between blood vessels and neurons, and astrocyte density is reduced in MDD. This study investigated the coverage of vessels by astrocyte endfeet in the prefrontal cortex in MDD.
Thirteen pairs of MDD and non-psychiatric control subjects were used for double immunofluorescent staining and confocal image analysis. Frozen sections of gray matter from orbitofrontal area 47 and white matter from the ventro-medial prefrontal cortex were examined. Astrocytic processes (labeled with antibodies for aquaporin-4, AQP4 or glial fibrillary acidic protein, GFAP) were co-localized with blood vessels (labeled with an antibody to collagen IV) to measure the coverage of vessel walls by astrocyte processes.
The coverage of blood vessels by endfeet of AQP4-immunoreactive (IR) astrocytes was significantly reduced by 50 percent in subjects with MDD as compared to controls (ANCOVA: F(1,23)=5.161, p=0.033). This difference was detected in orbitofrontal gray matter but not in white matter. Conversely, the coverage of vessels by GFAP-IR processes did not significantly differ between the groups.
A significant reduction in the coverage of gray matter vessels by AQP4-IR astrocyte processes in MDD suggests alterations in AQP4 functions such as regulation of water homeostasis, blood flow, glucose transport and metabolism, the blood brain barrier, glutamate turnover and synaptic plasticity.
PMCID: PMC3578083  PMID: 23146357
aquaporin-4; GFAP; glia; neurovascular unit; blood brain barrier; cerebrovascular disease
2.  New surgical modification of fascial closure following endovascular aortic pathology repair 
There are clear benefits of percutaneous versus open femoral access for endovascular aortic pathology repair. All closing devices commercially available are expensive. Surgical closure of the femoral artery risks potential prolonged wound healing and as a consequence longer hospital stay. Fascial closure is a technique that remains an interesting option.
To evaluate the efficacy of the surgical modification of hemostasis control after endovascular repair of aortic pathology.
Material and methods
One hundred sixteen common femoral arteries in a group of 58 patients underwent a minimally invasive procedure. Patients suffering from abdominal, thoracic aorta aneurysms, acute thoracic aorta type B dissections and traumatic aortic injury were treated.
A 1-year period of experience in fascial closure of 116 common femoral arteries was presented in the group of 58 patients undergoing endovascular interventions. Five intraoperative complications were observed and one late. Three primary failures were due to hemorrhage in three arteries, one required open repair and two additional compression after the procedure. Two cases of limb ischemia required surgical correction of artery closure. One limb ischemia was detected 4 weeks later, and was treated conservatively. At 1 year, 92 fascial closures (80%) were in the follow-up and 24 (20%) were lost to follow-up.
This new modification of fascial closure is a safe and cheap method of arterial closure following endovascular repair of selected aortic pathologies. The usage of two suture lines makes this procedure easy and quick. Fascial closure technique is comparable to other techniques in terms of success and complication rates.
PMCID: PMC3983539
endovascular procedure; fascial closure; closing device
3.  Crosslinking Protein Glutathionylation Mediated by O2-Arylated Bis-Diazeniumdiolate “Double JS-K” 
Chemical research in toxicology  2012;25(12):2670-2677.
Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, e.g., by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semi-quantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein’s thiol group are covalently bound via a crosslinking agent, rather than through a disulfide bond. This form of thiol crosslinking has been shown to occur and confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.
PMCID: PMC3524378  PMID: 23106594
4.  Co-transcriptional production of RNA–DNA hybrids for simultaneous release of multiple split functionalities 
Nucleic Acids Research  2013;42(3):2085-2097.
Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.
PMCID: PMC3919563  PMID: 24194608
5.  Activation of different split functionalities upon re-association of RNA-DNA hybrids 
Nature nanotechnology  2013;8(4):296-304.
Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications.
PMCID: PMC3618561  PMID: 23542902
6.  Listeria monocytogenes-Derived Listeriolysin O Has Pathogen-Associated Molecular Pattern-Like Properties Independent of Its Hemolytic Ability 
There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.
PMCID: PMC3535771  PMID: 23136118
7.  Structural modifications modulate stability of glutathione-activated arylated diazeniumdiolate prodrugs 
Bioorganic & medicinal chemistry  2012;20(9):3094-3099.
JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs’ biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound’s in vivo anti-tumor activity.
PMCID: PMC3590845  PMID: 22480849
Nitric oxide; Diazeniumdiolate prodrugs; JS-K; Glutathione; Anti-cancer agents
8.  Thiol Modification By Pharmacologically Active Agents of the Diazeniumdiolate Class 
Promising drug candidates of the diazeniumdiolate (NONOate) chemical family include several types of thiol modification among their mechanisms of action: 1) drugs designed to release nitric oxide (NO) on reaction with the thiol group of glutathione (GSH) arylate the GSH, a step that removes reducing equivalents from the cell; (2) a similar reaction of the drug with the thiol group of a protein changes its structure, leading to potentially impaired function and cell death; (3) the NO generated as a byproduct in the above reactions can undergo oxidation, leading to S-nitrosylation and S-glutathionylation; and (4) diazeniumdiolates can also generate nitroxyl, which reacts with thiol groups to form disulfides or sulfinamides.
PMCID: PMC3622254  PMID: 23585982
thiol; nitric oxide; diazeniumdiolate
9.  Diazeniumdiolated carbamates: A novel class of nitric oxide donors 
Bioorganic & medicinal chemistry  2012;20(6):2025-2029.
We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.
PMCID: PMC3298570  PMID: 22356735
Nitric oxide; Diazeniumdiolate prodrugs; Diazeniumdiolated carbamates; TOM protecting group; Intracellular nitric oxide
10.  Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance☆ 
Redox Biology  2013;1(1):115-124.
JS-K is a nitric oxide (NO)-releasing prodrug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.
Graphical abstract
► JS-K is a NO-releasing prodrug, with anticancer activity in vitro and in vivo. ► JS-K metabolism results in the arylation of cellular glutathione (GSH). ► Micromolar levels of JS-K in media significantly deplete the cellular GSH reserve. ► Deregulation of cells' redox balance is a key contributor to JS-K toxicity.
PMCID: PMC3757670  PMID: 24024144
ATF, activating transcription factor; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DCF-DA, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSH, glutathione; GSSG, glutathione disulfide (oxidized GSH); HBSS, Hank's balanced salt solution; IPA, Ingenuity Pathway Analysis; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate; LC/MS, liquid chromatography/mass spectrometry; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NO, nitric oxide; NSCLC, non-small cell lung cancer; PARP, poly (ADP-ribose) polymerase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK/JNK, stress activated protein kinase/c-jun N-terminal kinase.; Glutathione; Nitric oxide; Arylated diazeniumdiolate; Leukemia
11.  The Role of Mcl-1 in S. aureus-Induced Cytoprotection of Infected Macrophages 
Mediators of Inflammation  2013;2013:427021.
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.
PMCID: PMC3569898  PMID: 23431241
12.  Dose-Dependent Effects of Neonatal SSRI Exposure on Adult Behavior in the Rat 
Brain research  2011;1429:52-60.
Neonatal exposure to antidepressants produces lasting impairments in male sexual behavior. Although perturbation of the serotonin system during neonatal life has been implicated in the long-term behavioral effects of neonatal antidepressant exposure, dose-response studies were necessary to confirm that inhibition of the serotonin transporter during the neonatal period is sufficient to produce impairments in sexual behavior. Therefore, the present study examined the dose-response effects of neonatal citalopram exposure on sexual behavior. In addition, the effects of exposure on anxiety-related behavior were examined since alterations in this behavioral measure could affect sexual behavior. Male Long-Evans rats were injected subcutaneously with citalopram (CTM) in one of three doses (5, 10 or 20 mg/kg/day), or saline (SAL) in a volume of 0.1 ml twice daily (07:00 and 14:00h) from PN8 to PN21. The rats were tested as adults (>PN90) for anxiety-like behavior and exploration in the elevated plus maze test and sexual behavior.
Neonatal citalopram exposure produced persistent reductions in male sexual behavior characterized by significant dose-dependent reductions in the percentage of male rats displaying mounting as well as dose-dependent reductions in the number of mounts and mount latency. Neonatal citalopram exposure also produced significant dose-dependent linear trends for reductions in intromission and ejaculation behavior. However, neonatal SSRI exposure was not found to produce any effects on exploration or anxiety-like behavior in the elevated plus maze test. The present findings support the hypothesis that inhibition of the serotonin transporter during neonatal life by an SSRI is directly responsible for the long-term effects on male sexual behavior.
PMCID: PMC3228278  PMID: 22079319
Neonatal exposure; Citalopram; Sexual behavior; Anxiety
13.  Activation of the c-Jun N-terminal Kinase /Activating Transcription Factor 3 (ATF3) Pathway Characterizes Effective Arylated Diazeniumdiolate-Based Nitric Oxide-Releasing Anticancer Prodrugs 
Journal of medicinal chemistry  2011;54(22):7751-7758.
Improved therapies are needed for non-small cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)–releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against non-small cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue, and structural modification of these into more stable prodrugs. Compound 1 and its homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have anti-tumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.
PMCID: PMC3422893  PMID: 22003962
14.  Clinical efficacy of antazoline in rapid cardioversion of paroxysmal atrial fibrillation -- a protocol of a single center, randomized, double-blind, placebo-controlled study (the AnPAF Study) 
Trials  2012;13:162.
Rapid conversion of atrial fibrillation (AF) to sinus rhythm may be achieved by the administration of class IA, IC and III antiarrhythmic drugs or vernakalant hydrochloride. However, that treatment may be related to potential pro-arrhythmia, lack of efficacy or the exceptionally high cost of a compound used. Antazoline is a first generation antihistaminic agent with chinidin-like properties. When administered intravenously, antazoline exerts a strong antiarrhythmic effect on supraventricular arrhythmia, especially on AF, facilitating rapid conversion to sinus rhythm. Despite a relative lack of published data antazoline has been marketed in Poland and widely used in cardiology wards and emergency rooms for many years due to its efficacy, safety and rapid onset of action within minutes of administration.
A randomized, double blind, placebo-controlled, superiority clinical trial was designed to assess clinical efficacy of antazoline in rapid conversion of AF to sinus rhythm. Eligible patients will present AF lasting less than 43 hours, will be in stable cardio-pulmonary condition and will have no prior history of advanced heart failure or significant valvular disease. Long-term antiarrhythmic therapy is not considered an exclusion criterion. Subjects who fulfill selection criteria will be randomly assigned to receive intravenously either antazoline or placebo in divided doses and observed for 1.5 hours after conversion to sinus rhythm or after the last i.v. bolus. Primary end point will be the conversion of AF to sinus rhythm confirmed in an electrocardiogram (ECG) during the observation period. Secondary end points will be comprised of time to conversion and return of AF during the observation period. Special consideration will be given to the observation of any adverse events. A sample size of 80 patients was calculated based on the following assumptions: two-tailed test, a type I error of 0.01, a power of 90%, efficacy of placebo 5%, efficacy of antazoline 50% and 20% drop-out rate to fulfill the criteria of intention-to-treat analysis. Due to the presumed lack of statistical power, the secondary end points and safety endpoints will be considered exploratory.
Clinical trials registry, NCT01527279
PMCID: PMC3502525  PMID: 22967497
Antazoline; Atrial fibrillation; Pharmacological cardioversion; Efficacy; Safety; Randomized clinical trial
15.  First Universities Allied for Essential Medicines (UAEM) Neglected Diseases and Innovation Symposium 
Universities Allied for Essential Medicines organized its first Neglected Diseases and Innovation Symposium to address expanding roles of public sector research institutions in innovation in research and development of biomedical technologies for treatment of diseases, particularly neglected tropical diseases. Universities and other public research institutions are increasingly integrated into the pharmaceutical innovation system. Academic entities now routinely undertake robust high-throughput screening and medicinal chemistry research programs to identify lead compounds for small molecule drugs and novel drug targets. Furthermore, product development partnerships are emerging between academic institutions, non-profit entities, and biotechnology and pharmaceutical companies to create diagnostics, therapies, and vaccines for diseases of the poor. With not for profit mission statements, open access publishing standards, open source platforms for data sharing and collaboration, and a shift in focus to more translational research, universities and other public research institutions are well-placed to accelerate development of medical technologies, particularly for neglected tropical diseases.
PMCID: PMC3247111  PMID: 22232453
16.  The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline 
ACS medicinal chemistry letters  2010;1(8):386-389.
V-PYRRO/NO is a well studied nitric oxide (NO) prodrug which has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using 14C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in development of further NO prodrugs with increased permeability.
PMCID: PMC3013513  PMID: 21212855
Nitric oxide; prodrug; proline; transporter; PROLI/NO; V-PROLI/NO
17.  ‘Click’ Reaction in Conjunction with Diazeniumdiolate Chemistry: Developing High-Load Nitric Oxide Donors 
Organic letters  2010;12(19):4256-4259.
The use of Cu(I)-catalyzed ‘click’ reactions of alkyne-substituted diazeniumdiolate prodrugs with bis- and tetrakisazido compounds is described. The ‘click’ reaction for the bis-azide using CuSO4/Na-ascorbate predominantly gave the expected bis-triazole. However, CuI/diisopropylethylamine predominantly gave uncommon triazolo-triazole products as a result of oxidative coupling. Neither set of ‘click’ condition showed evidence of compromising the integrity of the diazeniumdiolate groups. The chemistry developed has applications in the synthesis of polyvalent and dendritic nitric oxide donors.
PMCID: PMC2945422  PMID: 20812718
18.  Deregulated Expression of Superoxide Dismutase-2 Correlates with Different Stages of Cervical Neoplasia 
Disease markers  2011;30(6):275-281.
Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer.
Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO).
Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining.
Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.
PMCID: PMC3825245  PMID: 21725155
Human papillomavirus; Tissue Microarray; Biomarker; Cervical Ne oplasia; Superoxide Dismutase-2
19.  Neonatal Antidepressant Exposure has Lasting Effects on Behavior and Serotonin Circuitry 
A significant fraction of infants born to mothers taking selective serotonin reuptake inhibitors (SSRIs) during late pregnancy display clear signs of antidepressant withdrawal indicating that these drugs can penetrate fetal brain in utero at biologically significant levels. Previous studies in rodents have demonstrated that early exposure to some antidepressants can result in persistent abnormalities in adult behavior and indices of monoaminergic activity. Here, we show that chronic neonatal (postnatal days 8–21) exposure to citalopram (5 mg/kg, twice daily, s.c.), a potent and highly selective SSRI, results in profound reductions in both the rate-limiting serotonin synthetic enzyme (tryptophan hydroxylase) in dorsal raphe and in serotonin transporter expression in cortex that persist into adulthood. Furthermore, neonatal exposure to citalopram produces selective changes in behavior in adult rats including increased locomotor activity and decreased sexual behavior similar to that previously reported for antidepressants that are nonselective monoamine transport inhibitors. These data indicate that the previously reported neurobehavioral effects of antidepressants are a consequence of their effects on the serotonin transporter. Moreover, these data argue that exposure to SSRIs at an early age can disrupt the normal maturation of the serotonin system and alter serotonin-dependent neuronal processes. It is not known whether this effect of SSRIs is paralleled in humans; however, these data suggest that in utero, exposure to SSRIs may have unforeseen long-term neurobehavioral consequences.
PMCID: PMC3118509  PMID: 16012532
neonatal exposure; citalopram; clomipramine; tryptophan hydroxylase; serotonin transporter; behavior
20.  Macrophage-dependent nitric oxide expression regulates tumor cell detachment and metastasis after IL-2/anti-CD40 immunotherapy 
The Journal of Experimental Medicine  2010;207(11):2455-2467.
Immunotherapy with IL-2 and anti-CD40 induces the expression of NOS2 in tumor-associated macrophages, and its expression is required for the inhibition of tumor metastasis.
Using an orthotopic model of renal cell carcinoma, we showed previously that IL-2/anti-CD40 immunotherapy resulted in synergistic anti-tumor responses, whereas IL-2 or α-CD40 alone mediated partial transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/α-CD40, but not IL-2 or α-CD40, induced significant nitric oxide synthase (NOS) 2 expression in tumor-associated macrophages. In control-treated mice (low NO), NOS2 inhibition reduced tumor burden. However, during immunotherapy (high NO), NOS2 inhibition or macrophage depletion reversed the ability of IL-2/α-CD40 treatment to reduce lung metastases but had no effect on primary tumor burden. Furthermore, IL-2/α-CD40 induced the IFN-γ– and NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with increases in tissue inhibitor of metalloproteinase (TIMP) 1 and E-cadherin expression within tumors. Finally, treatment of tumor-bearing mice with the NO donor JS-K significantly reduced metastases. These data differentiate the mechanism for primary anti-tumor effects of IL-2/α-CD40 immunotherapy, which are independent of NO, from the NO-dependent inhibition of metastases. Furthermore, reduced MMP9 activity implicates M1-polarized macrophages within the tumor microenvironment as critical components of therapeutic response. Our data demonstrate the mechanistic basis for IL-2/α-CD40–mediated control of metastases and suggest that the context-dependent application of NO donors may hold promise for prevention of metastatic disease.
PMCID: PMC2964582  PMID: 20921282
21.  Stabilization of the Nitric Oxide (NO) Prodrugs and Anti-Cancer Leads, PABA/NO and Double JS-K through Incorporation into PEG-Protected Nanoparticles 
Molecular pharmaceutics  2010;7(1):291.
Here we report the stabilization of the nitric oxide (NO) prodrugs and anti-cancer lead compounds, PABA/NO (O2-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and “Double JS-K” (1,5-bis{[1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato]-2,4-dinitrobenzene), through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.
PMCID: PMC2815019  PMID: 20000791
Nitric oxide; PABA/NO; glutathione; glutathione S-transferase; nanoparticles; formulation
22.  Glycosylated PROLI/NO Derivatives as Nitric Oxide Prodrugs 
Organic letters  2010;12(1):56-59.
GlcNAc-PROLI/NO prodrugs that are activated by N-acetylglucosaminidase to release nitric oxide (NO) are described. A classical acid-amine coupling is used to bi-functionalize these PROLI/NO prodrugs, which on activation generate up to 4 moles of NO, a peptide residue and an N-acetylglucosamine residue. Many of the prodrugs synthesized are efficient sources of intracellular NO.
PMCID: PMC2808138  PMID: 19954198
23.  Cancer immunotherapy targeting the HMW-MAA protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature 
Cancer research  2008;68(19):8066-8075.
The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are important for tumor angiogenesis. We generated a recombinant Listeria monocytogenes (Lm-LLO-HMW-MAA-C) that expresses and secretes a fragment of HMW-MAA (residues 2,160–2,258) fused to the first 441 residues of the listeriolysin O (LLO) protein. Immunization with Lm-LLO-HMW-MAA-C was able to impede the tumor growth of early established B16F10-HMW-MAA tumors in mice and both CD4+ and CD8+ T cells were required for therapeutic efficacy. Immune responses to a known HLA-A2 epitope present in the HMW-MAA2160–2258 fragment was detected in the HLA-A2/Kb transgenic mice immunized with Lm-LLO-HMW-MAA-C. Surprisingly, this vaccine also significantly impaired the in vivo growth of other tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. One hypothesis is that the vaccine could be targeting pericytes, which are important for tumor angiogenesis. In a breast tumor model, immunization with Lm-LLO-HMW-MAA-C caused CD8+ T-cell infiltration in the tumor stroma and a significant decrease in the number of pericytes in the tumor blood vessels. In conclusion, a Lm-based vaccine against HMW-MAA can trigger cell-mediated immune responses to this antigen that can target not only tumor cells but also pericytes in the tumor vasculature.
PMCID: PMC3004011  PMID: 18829565
24.  JS-K, an arylating nitric oxide (NO) donor, has synergistic anti-leukemic activity with cytarabine (ARA-C) 
Leukemia research  2009;33(11):1525-1529.
We have designed prodrugs that release NO on metabolism by Glutathione S-Transferases (GST). This design exploits the upregulation of GST in Acute Myeloid Leukemia (AML) cells. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl] diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. HL-60 myeloid leukemia cells were used for in vitro studies of the combination of JS-K with Daunorubicin (DAUNO), Cytarabine (ARA-C) or Etoposide (ETOP) using the median effect method to determine synergistic, antagonistic, or additive effects. Combinations of JS-K added simultaneously, 2 hours before or 2 hours after the other compounds were used. JS-K and DAUNO were antagonistic in all 3 drug sequences. JS-K and ETOP were also antagonistic but to a lesser degree. JS-K and ARA-C showed strong synergy. The combination index at the 50% fraction affected was 0.37 ± 0.23, 0.24 ± 0.27, and 0.15 ± 0.11 for simultaneous, JS-K first and ARA-C first additions, respectively. JS-K by itself induced DNA strand breaks at relatively high concentrations. However, at submicromolar concentrations, it significantly augmented ARA-C-induced DNA strand breaks. NMR spectroscopy revealed no evidence of chemical interaction between JS-K and the other chemotherapeutic agents. We conclude that ARA-C and JS-K have synergistic anti-leukemic activity and warrant further exploration in combination.
PMCID: PMC2756046  PMID: 19193435
25.  Neonatal citalopram exposure produces lasting changes in behavior which are reversed by adult imipramine treatment 
European journal of pharmacology  2006;532(3):265-269.
Neonatal exposure to antidepressants, including selective serotonin reuptake inhibitors such as citalopram, induces behavioral disturbances which persist in mature rats. These disturbances have been proposed to model the symptoms of endogenous depression. However, to date there is scant evidence for the predictive validity of any of these behaviors in response to adult antidepressant treatments. In order to directly assess the predictive validity of the early antidepressant exposure paradigm, the present study examined whether the behavioral abnormalities observed in adult animals exposed as neonates to citalopram can be reversed by adult antidepressant treatment with the prototypic antidepressant, imipramine. As noted earlier, neonatal citalopram exposure robustly increased locomotor activity and impaired male sexual behavior in adult rats. These behavioral changes were reversed following chronic adult imipramine treatment. No such reversal was observed in handled, saline treated rats. The present data support the hypothesis that some of the lasting behavioral abnormalities induced by early antidepressant exposure are sensitive to clinically relevant antidepressant treatments thus adding a measure of predictive validity to this paradigm as a model of these depressive symptoms.
PMCID: PMC2921633  PMID: 16483567
Neonatal treatment; Citalopram; Imipramine; Locomotor activity; Sexual behavior

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