High field structural MRI reveals specific episodic memory correlates in the subfields of the hippocampus.

The involvement of the hippocampus (HC) in episodic memory is well accepted; however it is unclear how each subfield within the HC contributes to memory function. Recent magnetic resonance imaging (MRI) studies suggest differential involvement of hippocampal subfields and subregions in episodic memory. However, most structural MRI studies have examined the HC subfields within a single subregion of the HC and used specialised experimental memory paradigms. The purpose of the present study was to determine the association between volumes of HC subfields throughout the entire HC structure and performance on standard neuropsychological memory tests in a young, healthy population. We recruited 34 healthy participants under the age of 50. MRI data was acquired with a fast spin echo (FSE) sequence yielding a 0.52×0.68×1.0 mm(3) native resolution. The HC subfields - the cornu ammonis 1-3 (CA), dentate gyrus (DG), and subiculum (SUB) - were segmented manually within three hippocampal subregions using a previously defined protocol. Participants were administered the Wechsler Memory Scale, 4th edition (WMS-IV) to assess performance in episodic memory using verbal (Logical Memory, LM) and visual (Designs, DE; visual-spatial memory, DE-Spatial; visual-content memory, DE-Content) memory subtests. Working memory subtests (Spatial Addition, SA; and Symbol Span, SSP) were included as well. Working memory was not associated with any HC volumes. Volumes of the DG were correlated with verbal memory (LM) and visual-spatial memory (DE-Spatial). Posterior CA volumes correlated with both visual-spatial and visual-object memory (DE-Spatial, DE-Content). In general, anterior subregion volumes (HC head) correlated with verbal memory, while some anterior and many posterior HC subregion volumes (body and tail) correlated with visual memory scores (DE-Spatial, DE-Content). In addition, while verbal memory showed left-lateralized associations with HC volumes, visual memory was associated with HC volumes bilaterally. This the first study to examine the associations between hippocampal subfield volumes across the entire hippocampal formation with performance in a set of standard memory tasks.

Platelets increase survival of adenocarcinoma cells challenged with anticancer drugs: mechanisms and implications for chemoresistance.

BACKGROUND AND PURPOSE: Cancer cells grow without the restraints of feedback control mechanisms, leading to increased cancer cell survival. The treatment of cancer is often complicated by the lack of response to chemotherapy leading to chemoresistance and persistent survival of tumour cells. In this work we studied the role of platelets in chemotherapy-induced cancer cell death and survival. EXPERIMENTAL APPROACH: Human adenocarcinoma cells, colonic (Caco-2) and ovarian (59 M) cells, were incubated with 5-fluorouracil (1-300 µg·mL(-1) ) or paclitaxel (1-200 µg·mL(-1) ) in the presence or absence of platelets (1.5 × 10(8) mL(-1) ) for 1, 24 or 72 h. Following incubation, cancer cells were harvested and cell survival/death was assayed using flow cytometry, Western blotting, real-time PCR, TaqMan® Gene Expression Assays and proteomics. KEY RESULTS: Human platelets increased the survival of colonic and ovarian adenocarcinoma cells treated with two standard anticancer drugs, 5-fluorouracil and paclitaxel. In the presence of platelets, cancer cells up-regulated anti-apoptotic and down-regulated pro-apoptotic genes, increased the number of cells in the synthesis of DNA and decreased the number in the quiescent phase, increased expression of cyclins, DNA repair proteins and MAPKs. The analysis of platelet-Caco-2 secretome demonstrated the release of the chemokine RANTES, thrombospondin-1, TGF-ß and clusterin. Finally, human recombinant RANTES and thrombospondin-1 improved survival of Caco-2 cells challenged with paclitaxel. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that platelets increase adenocarcinoma cells survival, proliferation and chemoresistance to standard anticancer drugs. Modulating cancer cell-platelet interactions may offer a new strategy to improve the efficacy of chemotherapy.

Nanoparticles: pharmacological and toxicological significance.

Nanoparticles are tiny materials (<1000 nm in size) that have specific physicochemical properties different to bulk materials of the same composition and such properties make them very attractive for commercial and medical development. However, nanoparticles can act on living cells at the nanolevel resulting not only in biologically desirable, but also in undesirable effects. In contrast to many efforts aimed at exploiting desirable properties of nanoparticles for medicine, there are limited attempts to evaluate potentially undesirable effects of these particles when administered intentionally for medical purposes. Therefore, there is a pressing need for careful consideration of benefits and side effects of the use of nanoparticles in medicine. This review article aims at providing a balanced update of these exciting pharmacological and potentially toxicological developments. The classes of nanoparticles, the current status of nanoparticle use in pharmacology and therapeutics, the demonstrated and potential toxicity of nanoparticles will be discussed.

Differential release of matrix metalloproteinase-9 and nitric oxide following infusion of endotoxin to human volunteers.

BACKGROUND: Roughly 400.000 cases of sepsis occur every year in the United States only and this is associated with a very high mortality. Bacterial lipopolysaccharide (LPS) triggers systemic inflammatory reactions in sepsis. However, down-stream cellular cascade initiated by LPS is still being elucidated. Nitric oxide (NO) and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) are known to be induced by LPS. We have investigated the release of NO, MMP-2 and MMP-9 following infusion of LPS to volunteers. METHODS: IPS (2 ng kg-1) was infused to 10 healthy volunteers. Before the experiments were started the subjects had an intravenous catheters placed. An electrocardiogram was also placed and monitored constantly. Body temperature was measured by ear thermometer every 10 min Venous blood was collected and cell-free plasma assayed for the presence of MMP-2 and MMP-9 using zymography and NO using HPLC assay for NO metabolites, nitrite and nitrate. RESULTS: The administration of LPS resulted in increased body temperature and tachycardia. Time-dependent release of MMP-9(30 fold increase from the baseline) peaking at 2 h following infusion of LPS was observed. LPS did not significantly modify the activity of MMP-2 (P > 0.05). Infusion of LPS did not significantly change the levels of nitrite and nitrate (from 60 +/- 11 to 67 +/- 10 micro m, P > 0.05). CONCLUSION: The release of MMP-9, but not MMP-2 or NO, is a sensitive index of endotoxaemia in humans. MMP-9 release may contribute to the pathogenesis of sepsis via its pro-inflammatory effects on the vasculature.

Pharmacological characteristics of solid-phase von Willebrand factor in human platelets.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.

Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin.

1. We have studied the effects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2. Washed platelet suspensions were obtained from human blood and the effects of HT-1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and flow cytometry. 3. Incubation of platelets with sVWF (1.2 microg ml(-1)) and HT-1080 cells (5 x 10(3) ml(-1)) resulted in a two-phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation. 4. TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 microM) and prostacyclin (PGI(2), 30 nM). 5. Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6. Pre-incubation of platelets with PGI(2) (30 nM) resulted in inhibition of sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 microM) did not affect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7. Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 microM) caused inhibition of activation of GPIIb/IIIa. 8. The inhibitors exerted no significant effects on TCIPA-mediated changes in GPIb. 9. Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these effects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10. Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI(2) differ from those of NO.

Cardiac surgery increases the activity of matrix metalloproteinases and nitric oxide synthase in human hearts.

OBJECTIVES: Heart function is variably impaired after cardiopulmonary bypass. We hypothesized that, similar to other myocardial injury states, cardiopulmonary bypass leads to enhanced activity of nitric oxide synthase and matrix metalloproteinases. METHODS: We obtained right atrial biopsy specimens and plasma samples at the onset and termination of cardiopulmonary bypass in 10 patients. Biopsy specimens were analyzed for nitric oxide synthase activity by using a citrulline assay, whereas plasma and tissue were analyzed for matrix metalloproteinase-9 and matrix metalloproteinase-2 activity by using zymography. Tissue inhibitor of metalloproteinase-4 was analyzed by means of Western blotting. The cellular expression of inducible nitric oxide, endothelial nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 was determined in right atrial biopsy samples from 3 additional patients by using the appropriate conjugated antibodies. RESULTS: Nitric oxide synthase activity increased from the beginning to the end of bypass (4.46 +/- 1.07 vs 16.77 +/- 4.86 pmol citrulline/mg of protein per minute, respectively; P =.018). Pro-matrix metalloproteinase-9 activity increased in hearts (199 +/- 41 vs 660 +/- 177 density units/mg protein; P =.008) and plasma (14.1 +/- 4.6 vs 52.2 +/- 5.9 density units/mg protein; P =.008). Pro-matrix metalloproteinase-2 activity increased in the heart (201 +/- 23 vs 310 +/- 35 density units/mg protein, P <.05) but not in plasma. Tissue inhibitor of metalloproteinase-4 expression in the heart decreased (1574 +/- 280 vs 864 +/- 153 density units, P =.014). CONCLUSIONS: Cardiopulmonary bypass activates enzymes mediating acute inflammation and organ injury (ie, nitric oxide synthase, matrix metalloproteinase-9, and matrix metalloproteinase-2). Decreased tissue inhibitor of metalloproteinase-4 expression allows relatively unopposed increases in matrix metalloproteinase tissue activity. We postulate that these changes play a role in the pathogenesis of heart dysfunction after bypass surgery.

Matrix metalloproteinase-2 in platelet adhesion to fibrinogen: interactions with nitric oxide.

BACKGROUND: Matrix metalloproteinase-2 (MMP-2) has been shown to activate a non-thromboxane, non-ADP pathway of platelet aggregation. In contrast, nitric oxide (NO) is known to inhibit platelet adhesion and aggregation. Therefore, we have studied the release of MMP-2 during platelet adhesion to fibrinogen, the effects of phenanthrolione, an MMP-2 inhibitor, on adhesion and the interactions of inhibitor with a NO donor, S-nitroso-N-acetyl-D, L-penicillamine (SNAP). MATERIAL AND METHODS: Human platelets were isolated from blood of healthy volunteers and platelet adhesion to fibrinogen-coated plates was studied by measuring thrombin-stimulated release of platelet a-granule constituent, platelet factor 4. In addition, the mode of action of phenanthroline and NO on platelets was investigated by assaying the levels of intraplatelet cyclic GMP. RESULTS: Thrombin-stimulated platelet adhesion to fibrinogen was associated with increased release of MMP-2 from platelets. Phenanthroline (0.1-100 KM) reduced platelet adhesion to fibrinogen. The adhesion was also inhibited by SNAP (0.1-100 KM), an effect abolished by 1H-[1,2,4] oxadiazolol [4,3,-a] quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylate cyclase. Co-administration of phenanthroline and SNAP resulted in a synergistic inhibition of platelet adhesion, an effect that was not associated with enhanced cyclic GMP generation by platelets. Furthermore, ODQ did not reverse the synergistic effect of these compounds on adhesion. CONCLUSIONS: 1. MMP-2 promotes platelet adhesion to fibrinogen. 2. Phenanthroline and NO synergize to inhibit platelet adhesion to fibrinogen acting through a cyclic GMP-independent mechanism(s).

Therapeutic effect of phenantroline in two rat models of inflammatory bowel disease.

BACKGROUND: Phenantroline is a zinc-chelator that inhibits biological activities of matrix metalloproteinases (MMPs). Over-expression of MMPs can accelerate tissue destruction and disrupt subsequent tissue repair. The effects of phenantroline in two rat models of inflammatory bowel disease (IBD) are evaluated: transmural colitis induced by trinitrobenzensulphonic acid (TNBS) and distal colitis caused by dextran sulphate sodium (DSS). METHODS: Transmural colitis was induced by TNBS in two groups of 15 rats each, and distal colitis was induced by DSS in two other groups of 15 rats each. Phenantroline was administered by oral gavage at 20 mg kg(-1) day(-1) to the test groups, whereas matched control groups received oral vehicle. On the last day of dosing, rats were subjected to intracolonic dialysis under anaesthesia for assessment of luminal eicosanoid release (PGE2, TXB2 and LTB4) and euthanized. Colons were removed and lesions were blindly scored according to macroscopic and histological scales. Myeloperoxidase (MPO) activity was measured in homogenates of colonic tissue. RESULTS: In the TNBS model, phenantroline treatment significantly reduced colonic strictures; in the DSS model, phenantroline significantly decreased scores of epithelial injury. In both models, the levels of PGE2, TXB2 and LTB4 and tissue MPO were not significantly altered. CONCLUSIONS: Although phenantroline did not modify the activity of inflammatory mediators, this compound substantially reduced intestinal injury associated with tissue remodelling.

Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells.

Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).

Inhibition of matrix metalloproteinase MMP-2 activates chloride current in human airway epithelial cells.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.

The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats.

The effects of nitric oxide (NO) and metalloproteinases (MMP-2 and MMP-9) in the pathogenesis of hyperoxia-induced lung damage in newborn rats were examined. Three-day-old rat pups were subjected to hyperoxia (> or = 95% O2) or room air for 7 and 14 days. Some animals were treated with NG-L-nitro-L-arginine methyl ester (L-NAME, 10 mg kg(-1), s.c., daily). Histology, morphometry, oedema, Ca2+-dependent and -independent NO synthase (NOS) activities, expression of NOS isoforms and the activities of MMP-2 and MMP-9 were measured in lungs of hyperoxic and control animals. Exposure of rats to hyperoxia for 7 days resulted in alveolar sac injury characterized by the presence of cellular debris, red cell extravasation and inflammatory infiltration with mononuclear cells. Lung water content, epithelial, smooth muscle layers and total airway thickness was similar to controls. In contrast, exposure of rats to hyperoxia for 14 days resulted in lung oedema, inflammation and epithelial proliferation. Hyperoxia caused a decrease in Ca2+-dependent NOS activity, an effect that was associated with increased expression of eNOS protein. In control rats, Ca2+-dependent NOS activity and expression of eNOS were reduced at 14 days. Hyperoxia caused 10 fold increase in the activity of Ca2+-independent NOS that remained significantly elevated after 14 days of exposure to hyperoxia. The activity of this enzyme was unchanged in control rats. In lungs of hyperoxic rats, the immunoblot showed time-dependent, biphasic expression (peak at 7 days) of iNOS. The profile of expression of iNOS in control rats was similar. The activities of MMPs were increased in lungs of hyperoxic animals. The L-NAME treatment of hyperoxic animals reduced lung oedema and epithelial proliferation, but enhanced the activities of MMPs. L-NAME exerted no significant effects in control rats. It is concluded that increased generation of NO contributes to the pathogenesis of hyperoxia-induced lung damage in newborn rats.

Nitric oxide activates chloride currents in human lung epithelial cells.

Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.

Tetrameres (Petrowimeres) striata in ducks.

Tetrameres (Petrowimeres) striata is an uncommon and incompletely described nematode from North American and Eurasian waterfowl. Specimens collected from mallards (Anas platyrhynchos) from the Playa Lakes Region (PLR) in western Texas (USA), the mottled duck (A. platyrhynchos fulvigula) in Florida (USA), the mallard in the Amur River Basin of eastern Russia and the blue-winged teal (Anas discors) from western Texas (USA) provide the basis for redescription of T. (P.) striata. This species is differentiated from the closely related T. (P.) zakharowi and T. (P.) galericulata by the size of the left spicule and the prominent cuff on the proximal end of the right spicule. In mallards from the PLR, one to several adult males and a single female occurred within distended mucosal glands or, more rarely, in cystic cavities in the submucosal tissue of the proventriculus; intensities ranged from one to seven nematodes per host (mean +/- SE = 2.9 +/- 0.8). Infected proventricular gland mucosae were compressed as a result of pressure atrophy by the large gravid females. In addition to nematodes, some lesions also contained necrotic debris with inflammatory cells and were surrounded by an intense inflammatory response of eosinophils, macrophages, lymphocytes, epithelioid cells and fibroblasts. Other lesions had little or no inflammatory response. Lesions in the submucosa were surrounded by a thin fibrous cyst with or without adjacent inflammatory cells.

Persistence of a recurrent group of intestinal helminth species in a coyote population from southern Texas.

The persistence and interrelationships of a recurrent group of intestinal helminth species in a coyote population from southern Texas were determined; data were collected at 4-yr intervals (1979, 1983, and 1987). The a priori expectation was that a defined recurrent group of helminth species persisted over time and that the respective helminth species were stable in terms of their abundances. The Fager index and subsequent recurrent group analysis indicated a small group of taxonomically unrelated recurring species of helminths that always demonstrated affinities with each other and occasionally with certain of the remaining species. Although there were differences across host subpopulations delineated by sex and age, temporal changes reflective of extrinsic environmental variables acting on certain helminth species appeared to have the greatest effect on recurrent group size and structure. Three of the 8 common intestinal helminth species (Ancylostoma caninum, Oncicola canis, and Alaria marcianae) occurred in all recurrent groups. Thus, there was temporal persistence of a small recurrent group of dominant helminth species that formed the basis of the intestinal helminth community in this coyote population.