Fluoroscein Isothiocyanate Research Articles

Synthesis and Binding of a Novel PSMA-specific Conjugate.

Prostate-specific membrane antigen (PSMA) is emerging as a target for treatment of castration-resistant prostate cancer (CRPC) while its up-regulated in the majority of CRPC tumors. The most common approach is targeted radionuclide therapy. The PSMA binding pharmacophore Glu-Urea-Lysine (GUL) and lysine were conjugated to oxidized dextran with reductive amination and subsequently labelled with fluorosceinisothiocyanate (FITC). Three prostate cancer cell lines were used for binding studies, 22Rv1 (PSMA positive), DU145 (PSMA negative) and PC3 (PSMA negative). Binding images were obtained by fluorescence microscopy. PDC binding was recorded on the 22Rv1 cell line while the negative cell lines showed no or slight background binding. PDC binding could be inhibited by pre-incubation with a molar excess of unlabelled PDC. This is a novel template for PSMA targeted CRPC therapy, either using cytostatics or radionuclides.

Alternating current impedance spectroscopic analysis of biofunctionalized vertically-aligned silica nanospring surface for biosensor applications

In this study, alternating current impedance spectroscopic analysis of the biofunctionalization process of a vertically-aligned (silica) nanosprings (VANS) surface is presented. The VANS surface is functionalized with a biotinylated immunoglobulin G (B-IgG) layer formed by physisorption of B-IgG from the solution phase. Bovine serum albumin passivation of the B-IgG layer reduces additional surface adsorption by blocking the potential sites of weak bond formation via electrostatic and hydrophobic interactions. As avidin acts as a receptor of biotinylated compounds, avidin conjugated glucose oxidase (Av-GOx) binds to the B-IgG layer via biotin. This avidin-biotin bond is a stable bond with high association affinity (Ka = 1015 M−1) that withstands wide variations in chemistry and pH. An IgG layer without biotin shows no binding to the Av-GOx, indicating that bonding is through the avidin-biotin interaction. Finally, fluoroscein iso-thiocyanate (FITC) labeled biotinylated bovine serum albumin (B-BSA) added to the Av-GOx surface is used to fluorescently label Av-GOx for fluorescent measurements that allow for the correlation of surface binding with impedance measurements. Modeling of impedance spectra measured after the addition of each biological solution indicates that the bimolecular layers behave as insulating layers. The impedance spectra for the VANS-based sensor are compared to simple parallel capacitor sensors, sans VANS, and serve as controls. VANS-based sensors exhibit a greater magnitude of change between successive bio-layers relative to the controls below 10 kHz. Changes in the magnitudes of the components of the VANS equivalent circuit indicate that the addition of biological layers changes the effective dielectric response of the VANS via the impediment of ionic motion and biomolecule polarization.

The modulation of intestinal endotoxin transport by different dietary fats

Intestinal derived endotoxin and the subsequent endotoxemia are considered to be major predisposing factors for diseases such as atherosclerosis, sepsis, obesity and diabetes. Therefore, the aim of this study was to assess the effects of dietary fat on intestinal endotoxin transport. We hypothesized that saturated fat (SF) would augment, while n‐3 fatty acids (FA) would attenuate endotoxin transport across the small intestine of pigs. Eleven pigs (three‐four weeks of age) were euthanized and jejunum segments were mounted in modified Ussing chambers. Segments were treated with either no oil (CON), 12.5% (v/v) vegetable oil (VO), DHA (DO), cod liver oil (CLO), coconut oil (CO) or olive oil (OO) in 20 mM bile acid. Ex vivo endotoxin transport was measured by the addition of fluoroscein isothiocyanate labeled lipopolysaccharide (FITC‐LPS, 20 μg/mL) and serial sampling of the mucosal and serosal chambers. Fluorescence was measured in a plate reader and an apparent permeability coefficient (Papp) was calculated using the surface area and transport rate. Compared to the CON, DO and CLO reduced the endotoxin Papp by 50% (P<0.01). However, versus the CON, CO increased the Papp by 60% (P=0.008). OO and VO Papp were not different from the CON. Overall, these data indicate that fat high in n‐3 FA can attenuate intestinal endotoxin transport, while SF augments endotoxin transport. This work was supported by the USDA and NWRC.

High Mucosal Serotonin Availability in Neonatal Guinea Pig Ileum Is Associated With Low Serotonin Transporter Expression

5-Hydroxytryptamine (5-HT) is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterochromaffin cells (EC), which release 5-HT, has not been studied in neonates. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs. 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and high-performance liquid chromatography with electrochemical detection. An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface, and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 microM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs adult tissues. There was no difference in 5-HT content between neonates and adults but 5-hydroxyindole acetic acid/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number, but EC cells were found in the crypts in neonatal and along the villi in adult tissues. SERT expression is low in neonates, and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may be important for development of neurohumoral control of intestinal motor reflexes.

Effects of delta-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro

To investigate effects of delta-9-tetrahydrocannabinol (THC) on human sperm function in vitro. Laboratory analysis of sperm motility after exposure to THC using computer-assisted semen analysis and acrosome reaction by fluoroscein isothiocyanate-labeled peanut agglutinin staining. An assisted reproductive technology unit. Seventy-eight male patients. Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with THC at concentrations equivalent to therapeutic (0.032 microM) and recreational (0.32 and 4.8 microM) plasma levels at 37 degrees C for 3 h. Sperm motility and spontaneous and induced acrosome reactions. Percentage progressive motility was decreased dose dependently in the 90% fraction (by 2%-21%; P<.05; P<.001). The 45% fraction showed a greater decrease in percentage progressive motility (by 28% at 0.032 microM; 56% at 4.8 microM; P=.004 and P=.01 res). Straight line velocity and the average path velocity also were reduced (by 10%, in the 90% LAYER) in both fractions. Spontaneous acrosome reactions were reduced in the 90% (17% at 0.032 microM, 35% at 4.8 microM P=.004 and P<.001 resp) and more markedly in the 45% fractions (17%-35%; P<.001). When the acrosome reaction was artificially induced (90% fraction) by A23187, THC (4.8 microM) resulted in a 57% inhibition (P<.001). The use of THC as a recreational drug may adversely affect male fertility.

The Ig-ITIM superfamily member PECAM-1 regulates the “outside-in” signaling properties of integrin αIIbβ3 in platelets

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.

CD66 Expression on Malignant and Normal Plasma Cells: A Potential Target for Therapy.

CD66, a member of the carcinoembryonic antigen family, is known to be expressed on cells of myeloid and monocytic origin and has also been demonstrated on blasts from patients with B-lineage acute lymphoblastic leukaemia. An analysis of CD66 expression has been undertaken in bone marrow samples from patients with plasma cell disorders. Diagnostic bone marrow aspirate samples from 53 patients with multiple myeloma or monoclonal gammopathy were examined by multiparametric flow cytometry in order to demonstrate and quantitate plasma cells. Samples were analysed initially using a primary screening panel of antibodies including a combination of antiCD19 fluoroscein isothiocyanate (FITC)(Pharminogen), antiCD5 phycoerythrin (PE)(in-house), antiCD45 peridin chlorophyll protein (PerCP)(Becton Dickinson) and antiCD38 allophycocyanin (APC)(Phar). Samples shown to contain a CD45 negative, CD19 negative, CD38 positive population, consistent with the presence of plasma cells were then examined with a myeloma panel, including antiCD66. Samples from 41 patients were analysed using antiCD66 naked antibody (TheraPharm, GmBH) double-layered with sheep anti-mouse IgG F(ab')2 fragment conjugated to FITC, antiCD138 PE, antiCD38 APC and antiCD45 PerCP. The correlation between CD38/CD138 and CD38/CD66 dual expression was 0.997. A further 13 samples were analysed using a commercial antiCD66 antibody conjugated to FITC (Dako). Again the correlation between CD38/CD138 and CD38/CD66 dual expression was 0.990. All patients apart from one, with morphologically detectable plasma cells, co-expressed CD66 with CD38. One patient with highly plasmablastic morphology did not express either CD38/138 or CD38/66 on plasma cells.The bone marrow of a normal individual was also examined and found to contain plasma cells which co-expressed CD19, CD138, CD38 and CD66. Plasma cells express CD66 in almost all patients with plasma cell disorders and expression has also been demonstrated on plasma cells in a normal individual. CD66 is therefore an attractive target for immunotherapy. A clinical trial using anti-CD66 targeted radiotherapy as part of the conditioning regimen for patients undergoing autologous or allogeneic stem cell transplantation, as therapy for multiple myeloma, is currently being conducted at our institution.

Epithelial internalization of superparamagnetic nanoparticles and response to external magnetic field

Superparamagnetic magnetite nanoparticles (MNP) coated with silica were synthesized and chronically implanted into the middle ear epithelial tissues of a guinea pig model (n=16) for the generation of force by an external magnetic field. In vivo limitations of biocompatibility include particle morphology, size distribution, composition and mode of internalization. Synthesis of MNP was performed using a modified precipitation technique and they were characterized by transmission electron microscopy, X-ray diffractometry and energy dispersive spectroscopy, which verified size distribution, composition and silica encapsulation. The mechanism for internalizing 16±2.3nm diameter MNP was likely endocytosis, enhanced by magnetically force. Using sterile technique, middle ear epithelia of tympanic membrane or ossicles was exposed and a suspension of particles with fluoroscein isothiocyanate (FITC) label applied to the surface. A rare earth, NdFeBo magnet (0.35T) placed under the animal, was used to pull the MNP into the tissue. After 8 days, following euthanasia, tissues were harvested and confocal scanning laser interferometry was used to verify intracellular MNP. Displacements of the osscicular chain in response to an external sinusoidal electromagnetic field were also measured using laser Doppler interferometry. We showed for the first time a physiologically relevant, biomechanical function, produced by MNP responding to a magnetic field.

Utility of endogenous creatinine clearance as a measure of renal function in mice

The use of endogenous plasma creatinine levels and creatinine clearance as a tool to evaluate renal function in mice has come under scrutiny as prior studies have reported that the Jaffé alkaline picrate method grossly overestimates true plasma creatinine in mice. As members of the NIDDK Animal Models of Diabetic Complications Consortium (AMDCC), we evaluated the performance and feasibility of an alternative high-performance liquid chromatography (HPLC)-based method for standard determination of plasma creatinine and creatinine clearance in mice. Our purpose was to develop a simple method that provides a reliable, reproducible, and sensitive assay for small volumes (<25 microL) of mouse plasma and sera. We compared creatinine clearance measured by HPLC with the Jaffé method and HPLC creatinine clearance with inulin clearance [fluoroscein isothiocyanate (FITC) inulin in an osmotic pump implanted in mouse] in C57BL/6J mice. Different groups of mice underwent either one of two protocols. Protocol A included dietary intervention with normal, low salt plus enalapril, or high salt. Protocol B induced diabetes using streptozotocin. First, mean plasma creatinine levels were significantly lower (P < 0.0001) by HPLC (0.128 +/- 0.026 mg/dL) vs. Jaffé (0.4 +/- 0.12 mg/dL) for mice on a normal diet. Urine creatinine concentrations measured by HPLC were 10% lower than by Jaffé (P < 0.01). Second, mean creatinine clearance by HPLC for mice on a normal diet was 255 +/- 68 microL/min. Mice on low salt diet plus enalapril had reduced creatinine clearance (72.8 +/- 24.2 microL/min) while mice on high salt diet had an elevated creatinine clearance (355 +/- 105 microL/min). Third, diabetic mice (19 to 24 weeks of diabetes) exhibited hyperfiltration as creatinine clearance was 524 +/- 214 microL/min whereas nondiabetic age/gender-matched mice showed a mean creatinine clearance of 206 +/- 41 microL/min. Finally, significant correlation was demonstrated for creatinine clearance by HPLC vs. inulin clearance (R= 0.643; P < 0.001). HPLC is highly accurate, much more sensitive and specific than the Jaffé method for plasma creatinine measurements in mice. Creatinine clearance in mice measured by HPLC reflects changes in renal function induced by diet and diabetes.

9-cis-retinoic acid during in vitro maturation improves development of the bovine oocyte and increases midkine but not IGF-I expression in cumulus-granulosa cells.

The isomer 9-cis of retinoic acid (9-cis-RA) exerts a beneficial effect on bovine in vitro development when added to in vitro maturation (IVM) culture. In the present work, 9-cis-RA 5 nM was found to be stimulatory as opposed to 500 nM (toxic). Cumulus-oocyte complexes (COCs) were treated with the found physiological dose 9-cis-RA 5 nM, and the next determinations performed: (1) relative expression of midkine (MK) and IGF-I, by reverse transcriptase-polymerase chain reaction (RT-PCR), in cumulus-granulosa cells detached from oocytes; (2) cytoplasmic granular migration, by labeling of oocytes with fluoroscein isothiocyanate lectins; and (3) in vitro survival of blastocysts after vitrification and warming. Gene expression of MK was enhanced by 9-cis-RA, but not by 1% ethanol (vehicle). However, we did not detect IGF-I expression, both in dependence on or in the absence of 9-cis-RA acting on cumulus-granulosa cells. The ability of vitrified blastocysts to survive in vitro was not improved by 9-cis-RA. Nevertheless, since only blastocysts obtained from oocytes matured with serum survived, more factors should be considered when evaluating cryopreservation survival. The complete granular migration observed in oocytes matured with 9-cis-RA anticipates the gain in developmental competence of the oocyte, being MK probably involved in this beneficial effect.

Assay of tracheal pepsin as a marker of reflux aspiration.

Aspiration of gastric contents is a relatively common cause of acute and chronic pulmonary disease. However, a reliable method of diagnosing recurrent aspiration is currently lacking. The aim of this study was to determine whether the presence of gastric pepsin in tracheal aspirates of infants and children might be used as a reliable marker of the microaspiration of refluxed gastric contents. Ninety-eight children undergoing general anesthesia and tracheal intubation participated in the study. Sixty-four of 98 children underwent endoscopy for clinically significant gastroesophageal reflux. Thirty-four children from routine operative lists were nonreflux controls. These two groups were further subdivided based on the presence or absence of associated respiratory symptoms. After endotracheal intubation, tracheal aspirates were obtained and subsequently assayed for gastric pepsin using a fluoroscein isothiocyanate casein. Pepsin was detected in 7 of 27 children with reflux symptoms alone and in 7 of 8 of those with chronic respiratory symptoms. In addition, pepsin was present in 31 of 37 children with a history of both reflux and chronic respiratory symptoms. Tracheal pepsin was not detected in any of the 26 children without gastroesophageal reflux or respiratory symptoms. Tracheal pepsin was found significantly more frequently in children with reflux symptoms than in those without, particularly in children with both reflux and respiratory problems. Tracheal pepsin assay as a reliable marker of gastroesophageal reflux aspiration.

Assay of Tracheal Pepsin as a Marker of Reflux Aspiration

ABSTRACTObjectivesAspiration of gastric contents is a relatively common cause of acute and chronic pulmonary disease. However, a reliable method of diagnosing recurrent aspiration is currently lacking. The aim of this study was to determine whether the presence of gastric pepsin in tracheal aspirates of infants and children might be used as a reliable marker of the microaspiration of refluxed gastric contents.MethodsNinety‐eight children undergoing general anesthesia and tracheal intubation participated in the study. Sixty‐four of 98 children underwent endoscopy for clinically significant gastroesophageal reflux. Thirty‐four children from routine operative lists were nonreflux controls. These two groups were further subdivided based on the presence or absence of associated respiratory symptoms. After endotracheal intubation, tracheal aspirates were obtained and subsequently assayed for gastric pepsin using a fluoroscein isothiocyanate casein.ResultsPepsin was detected in 7 of 27 children with reflux symptoms alone and in 7 of 8 of those with chronic respiratory symptoms. In addition, pepsin was present in 31 of 37 children with a history of both reflux and chronic respiratory symptoms. Tracheal pepsin was not detected in any of the 26 children without gastroesophageal reflux or respiratory symptoms. Tracheal pepsin was found significantly more frequently in children with reflux symptoms than in those without, particularly in children with both reflux and respiratory problems.ConclusionTracheal pepsin assay as a reliable marker of gastroesophageal reflux aspiration.

Ingestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference.

RNA interference is of value in determining gene function in many organisms. Plant parasitic nematodes are refractory to microinjection as a means of introducing RNA and do not show any oral uptake until they are within plants. We have used octopamine to stimulate uptake by preparasitic second stage juveniles of two cyst nematodes, Heterodera glycines and Globodera pallida. This new technique was used to facilitate uptake of double stranded RNA (dsRNA) together with fluoroscein isothiocyanate as a visual marker. Targeting cysteine proteinases did not reduce the number of parasites but caused a shift from the normal female/male ratio of 3:1 to 1:1 by 14 days postinfection (dpi). Exposure of H. glycines to dsRNA corresponding to a newly characterized protein with homology to C-type lectins did not affect sexual fate, but 41% fewer parasites were recovered from the plants. As expected, treatment with dsRNA corresponding to the major sperm protein (MSP) had no effect on either parasite development or sexual fate over 14 days. Northern analysis showed lower transcript abundance for the two targeted mRNAs that occur in J2, plus a later inhibition for MSP transcripts when males developed sperm at 15 dpi. These findings establish a procedure for RNAi of plant parasitic nematodes.

Short exposure of maturing, bone marrow-derived dendritic cells to norepinephrine: impact on kinetics of cytokine production and Th development

The information gathered by dendritic cells (DC) during the innate immune response to a pathogen is determinant for the type of adaptive response. Here we show that short-term (3 h) exposure of bone marrow-derived DC to norepinephrine (NE), at the beginning of lipopolysaccharide (LPS) or keyhole limpet hemocyanin (KLH) stimulation hampers IL-12 production and increases IL-10 release. The NE effect was mediated by both beta- and alpha2-adrenergic receptors. The capacity of NE-exposed DC to produce IL-12 upon CD40 cross-linking as well as to stimulate allogeneic T-helper (Th) lymphocytes was reduced. Adoptive transfer of NE-exposed DC induced a Th2 slanted response in vivo. Thus, a brief NE exposure of antigen-stimulated DC seems to limit their Th1 polarizing properties. Noteworthy, the ganglionic blocker pentolinium administered in mice before skin sensitization with fluoroscein isothiocyanate (FITC) could increase the Th1-type response in the draining lymph nodes. Our results suggest that the extent of Th differentiation in the response to an antigen might be influenced by the local sympathetic nervous activity in the early phase of dendritic cell stimulation.

Use of lectin binding characteristics to identify gastrointestinal parasite eggs in faeces

Identification of the presence and abundance of species is important when choosing therapies and control strategies for internal parasitism of livestock. Here we examine lectin binding characteristics of eggs isolated from sheep faeces as a means for identifying the parasite genera contributing to infection. The intensity of lectin staining varied with incubation time, incubation volume, concentration of lectin and concentration of eggs. Formalin fixed eggs had greater autofluorescence but exhibited the same lectin staining pattern as fresh eggs. The stage of egg development did not influence staining. Eggs from Haemonchus contortus, H. placei, Trichostrongylus colubriformis, T. vitrinus, Ostertagia circumcincta, Nematodirus spathiger and the cestode Monezia expansa were incubated with a panel of fluoroscein isothiocyanate (FITC)-labelled lectins. Lectin binding exhibited a genus specific pattern. Haemonchus spp. stained strongly positive with peanut agglutinin (PNA), and were positive for concanavalin A (ConA), Ricinus communis agglutinin (RCA) and Maclura pomifera lectin (MPA). Trichostrongylus spp. were PNA−, ConA−, RCA− and strongly MPA+. O. circumcincta were weakly positive for PNA, MPA, ConA and negative for RCA. N. spathiger were weakly positive for the four lectins, and M. expansa were weakly positive for PNA, RCA and MPA and were strongly ConA+. The genus specificity of lectin staining was used to identify the presence of Trichostrongylus and Haemonchus eggs in faeces from sheep with mixed field infections, however correspondence between lectin staining and larval differentiation for identifying a low prevalence of Ostertagia in the field infection was poor. Refinements in methods for rapid egg isolation may improve egg differentiation on the basis of lectin staining, which could be undertaken by flow cytometry or microscopy.

L-selectin expression on thymic emigrants defines two distinct tissue-migration pathways.

We have studied the appearance and phenotype of recent thymic emigrants in blood, spleen and lymph nodes (LN) of neonatal lambs. Using in situ labelling of thymocytes with fluoroscein isothiocyanate (FITC), we examined the expression of the LN homing receptor L-selectin on alphabeta and gammadelta subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and gammadelta T-cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and gammadelta TCR+ emigrants while LN were enriched in CD4+ emigrants. There were also marked differences in the expression of L-selectin by emigrants homing to spleen compared with those homing to lymph nodes. While the majority of thymic emigrants in LN expressed L-selectin, considerably fewer emigrants in spleen were L-selectin+. The presence of large numbers of CD8+ L-selectin- and gammadelta TCR+ L-selectin- thymic emigrants homing to spleen raises the possibility that unique homing receptor specificities underpin the migration of T cells to spleen as distinct from lymph nodes.

Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact!

Myoglobin has been suggested to be a potential inhibitor of endogenous muscle proteases as different as cathepsin B, cathepsin L, cathepsin H and calpains all being supposed to be important in post-mortem muscle. The present work aimed at verifying the ability of myoglobin and its prosthetic group, hemin, to inhibit a series of endopeptidases including papain, cathepsin B, trypsin, calpains as well as two activities of the 20S proteasome. The conclusion of the present work was that inhibition of proteolytic activities of endopeptidases by myoglobin is an artifact. This was based on the following evidences: (1) a similar extent of inhibition was observed for all proteases tested whether myoglobin or hemin were added before starting the reaction or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assayed with non labeled casein as substrate and the activity expressed as the increase in the absorbency at 280 nm of the TCA soluble protein fragments. 1 Abbreviations: AMC, 7-amino-4-methyl coumaryl-amide; Boc, t-butyloxycarbonyl; DMSO, dimethyl-sulfoxyde; EDTA, ethylene-diamine-tetraacetic acid (tetrasodium salt); FITC, fluoroscein isothiocyanate; NAD, nicotinamide adenine dinucleotide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; Suc, succinyl; TCA, trichloro-acetic acid; Z, benzyloxycarbonyl. 1

Characterization and reduction of ischemia/reperfusion injury after experimental pancreas transplantation

Reperfusion injury after pancreas transplantation is a cause of early graft pancreatitis. The aim of this study was to quantify pancreatic microcirculation after pancreas transplantation in correlation with cold ischemia time. In a second step the effect of N-acetylcysteine on reperfusion damage was tested. Pancreas transplantation was performed in three different groups of male Lewis rats. Groups 1 and 2 received no special treatment. Cold ischemia time was 1.5 hours in group 1 and 16 hours in groups 2 and 3. In group 3 donor and recipient were both treated with N-acetylcysteine (300 mg/kg) 1.5 hours after reperfusion graft microcirculation was quantified by means of intravital microscopy. Rhodamine-labeled leukocytes, fluoroscein isothiocyanate—labeled erythrocytes, and fluoroscein isothiocyanate—albumin were used as fluorochromes. After a cold ischemia time of 16 hours, functional capillary density, erythrocyte velocity, and leukocyte-endothelium interaction were reduced significantly compared to a cold ischemia time of 1.5 hours ( P <0.05). After 16 hours of cold ischemia, treatment with N-acetylcysteine improved all of these parameters ( P ≤0.05). Ischemia/reperfusion injury after experimental pancreas transplantation is characterized by a disturbance of the pancreatic microcirculation exhibiting a correlation with the duration of cold ischemia. Treatment of donor and recipient with N-acetylcysteine resulted in prevention of cold ischemia—induced microcirculatory disturbance.

Characterization of Endotoxin and Cationic Liposome Interaction

The objective of this study was to characterize the interaction of endotoxin with cationic liposomes used in nonviral gene delivery. Endotoxin–cationic liposome interaction was characterized using fluorescent anisotropy, and the Limulus amebocyte lysate (LAL) assay. Cellular toxicity of endotoxin–cationic liposome complex was examined using a dimethylthiazol diphenyltetrazolium bromide (MTT) assay. The effect of endotoxin on the lipid–DNA complex and subsequent transfection into COS-1 cells was also examined. A competitive interaction occurred between fluoroscein isothiocyanate (FITC)-labeled endotoxin and plasmid DNA for binding dioleoyl glycero trimethylammonium propane:dioleoyl glycero phosphoethanolamine (DOTAP:DOPE) liposomes using fluorescent anisotropy techniques. The LAL assay demonstrated no change in endotoxin activity upon interaction with liposomes. No loss of COS cell viability was detected via the MTT assay during a 5-hr exposure to endotoxin. Transient transfection studies indicate that increasing levels of endotoxin lowered activity more than 90% at 50,000 endotoxin units (EU)/ml. Endotoxin and cationic liposomes interact mainly by an electrostatic attraction. Endotoxin contamination can potentially impact transfection efficiency via competition with plasmid DNA for cationic liposome binding by increasing transfection variability at 50 EU/ml, a concentration of endotoxin contamination that can occur with small-scale plasmid preparations used for in vitro cell transfections, but would not be expected with typical GLP or GMP preparations used in clinical studies.

Prostaglandin and fatty acid modulation of Escherichia coli O157 phagocytosis by human monocytic cells.

Phagocytosis by human monocytes is an important primary survival mechanism particularly during bacterial infection. However, the processes that control the events and mediators involved in the activation of monocytes and their impact on the phagocytosis of bacteria are poorly understood. The effect of bacterial endotoxin, interleukin-1 beta (IL-1 beta), fatty acids and prostaglandin E2 (PGE2) on the phagocytosis of fluoroscein isothiocyanate (FITC)-labelled Escherichia coli (O157) by human blood monocytes and U937 cells was studied by flow cytometry. Endotoxin increased the phagocytosis of labelled bacteria by both monocytes and U937 cells. IL-1 beta and the polyunsaturated fatty acids; dihomo-gamma-linolenic and arachidonic acids also increased the phagocytic activity of both monocytes and U937 cells. In contrast, PGE2 suppressed phagocytosis in a concentration-dependent manner. The cyclo-oxygenase inhibitor, ketoprofen, further enhanced the increased phagocytic activity in the presence of endotoxin and interleukin-1 (IL-1) indicating suppression by endogenous prostaglandins. This was confirmed by the data which showed that lipopolysaccharide (LPS) and IL-1 increased PGE2 release and ketoprofen inhibited release. Endotoxin and fatty acids increased IL-1 beta release also, whereas PGE2 inhibited release. The data suggest that phagocytic activity may be linked to changes in IL-1 levels. The data presented in this study also suggest that monocyte phagocytosis in the course of bacterial infection would be altered during pathophysiological events which result in elevation of extracellular fatty acids.