Schistosoma mansoni: Detection and characterization of schistosome derived antigens by inhibition enzyme-linked immunosorbent assay (IELISA) utilizing monoclonal antibodies

Monoclonal antibodies were used in an inhibition enzymelinked immunosorbent assay (IELISA) to detect a variety of schistosome derived antigens. Preparations were obtained from various stages ofSchistosoma mansoni and from the eggs ofS. japonicum. Using appropriate titers of monoclonal antibodies it was possible to detect less than 0.01 g/ml of schistosome antigens. The sensitivity of IELISA was dependent upon the type and concentration of the monoclonal antibody used, as well as upon the source of the antigens. Specificity studies showed that some of the monoclonal antibodies recognized species specific antigenic determinants, while others reacted against genus specific antigens. Furthermore, certain antibodies interacted with antigens which were neither genus or species specific. Fractions ofS. mansoni andS. japonicum egg antigen extracts, which have been previously considered to be relatively pure, were compared using certain monoclonal antibodies. The results indicate that these fractions are very heterogenous with regard to the unique antigenic specificities. For example, most antigenic determinants in crude soluble egg antigen are not retained on concanavalin-A lectin affinity columns and major serologic antigens have more than one determinant with different distributions. The expression of these antigenic determinants appears to be a function of both the concentration of the specific antigen and the mode of expression of the moiety within the antigenic matrix.     

Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

Cardiovascular Drugs and Therapy - Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent...     

Role of Oxidative Stress in a Rat Model of Radiation-Induced Erectile Dysfunction

IntroductionChronic oxidative stress is one of the major factors playing an important role in radiation-induced normal tissue injury. However, the role of oxidative stress in radiation-induced erectile dysfunction (ED) has not been fully investigated.AimsTo investigate role of oxidative stress after prostate-confined irradiation in a rat model of radiation-induced ED.MethodsFifty-four young adult male rats (10–12 weeks of age) were divided into age-matched sham radiotherapy (RT) and RT groups. Irradiated animals received prostate-confined radiation in a single 20 Gy fraction.Main Outcome MeasuresIntracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation were conducted at 2, 4, and 9 weeks following RT. The protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox4 and gp91^phox), markers of oxidative DNA damage (8-hydroxy-2′-deoxyguanosine [8-OHdG]), lipid peroxidation (4-hydroxynonenal [4HNE]), and inflammatory response including inducible nitric oxide synthase, macrophage activation (ED-1), and nitrotyrosine, and endogenous antioxidant defense by nuclear factor erythroid 2-related factor (Nrf2) were evaluated in irradiated prostate tissue and corpora cavernosa (CC). In addition, we investigated the relationships between results of ICP/mean arterial pressure (MAP) ratios and expression level of oxidative stress markers.ResultsIn the RT group, hemodynamic functional studies demonstrated a significant time-dependent decrease in ICP. Increased expression of Nox4, gp91^phox, 8-OHdG, and 4HNE were observed in the prostate and CC after RT. Similarly, expressions of inflammatory markers were significantly increased. There was a trend for increased Nrf2 after 4 weeks. ICP/MAP ratio negatively correlated with higher expression level of oxidative markers.ConclusionsNADPH oxidase activation and chronic oxidative stress were observed in irradiated prostate tissue and CC, which correlated with lower ICP/MAP ratio. Persistent inflammatory responses were also found in both tissues after RT. These findings suggest that oxidative stress plays a crucial role in the development of radiation-induced ED. Kimura M, Rabbani ZN, Zodda AR, Yan H, Jackson IL, Polascik TJ, Donatucci CF, Moul JW, Vujaskovic Z, and Koontz BF. Role of oxidative stress in a rat model of radiation-induced erectile dysfunction. J Sex Med 2012;9:1535–1549.     

Liquid gentamicin in bone cement: a laboratory study of a potentially more cost-effective cement spacer

BACKGROUND: Liquid gentamicin is approved by the Food and Drug Administration, is readily available, and is much less costly than tobramycin, the more commonly used antibiotic in cement spacers. The purpose of the present study was to test the mechanical properties, elution characteristics, and antibacterial activity of liquid gentamicin in an acrylic bone cement spacer. METHODS: Standardized specimens consisting of Palacos cement combined with liquid gentamicin, powdered tobramycin, or no antibiotic were fashioned, and the three groups were tested with regard to compressive and tensile strength, elution characteristics (with use of radioimmunoassays), and antibacterial activity (with use of bioassays). RESULTS: The ultimate compression strength decreased by 49% (from 64.65 +/- 3.89 MPa to 32.96 +/- 3.33 MPa) and the ultimate tension strength decreased by 46% (from 35.85 +/- 2.97 MPa to 19.20 +/- 0.36 MPa) when the specimens containing 480 mg of liquid gentamicin were compared with the controls. The addition of tobramycin had no significant effect compared with the controls. The majority of gentamicin was released from the cement during the initial twenty-four hours (mean concentration, 26.4 mcg/mL). The mean concentrations at three and six weeks were 4.15 and 0.65 mcg/mL, respectively. The bioassays confirmed the bactericidal activity of the gentamicin released from the cement. CONCLUSIONS: Liquid gentamicin in bone cement is potent and bactericidal. Although the mechanical properties of the cement are significantly diminished by the addition of liquid gentamicin, the temporary nature of the cement spacer makes its use potentially worthwhile given the substantial cost savings to the hospital and the patient.     

Effect of pH on the formation of Tc(NaBH4)-MDP radiopharmaceutical analogues

Anion exchange high performance liquid chromatography has been employed to separate components of 99mTc(NaBH4)-MDP formulations prepared as a function of pH, the presence or absence of 99Tc carrier, and the presence or absence of air. The formulation pH is an effective variable for controlling the generation and interconversion of 99mTc-MDP components, and for allowing the preparation of large quantities of specific 99mTc-MDP complexes for biological evaluation.     

Severe malaria presenting to the ED: A collaborative approach utilizing exchange transfusion and artesunate

We describe the case of a previously healthy 33 year-old male pilot recently arrived to the United States from Africa. The patient presented to our ED febrile and disoriented, with projectile coffee-ground emesis. He was later found to have severe malaria and cerebral parasitemia. Due to the severity of his illness, the patient received the anti-malarial medication Artesunate as well as several exchange transfusions. Within 48 h his parasitic load was reduced from 42% to 0.4%. The following is an account of a collaborative effort that spans the specialties of emergency medicine, infectious disease, and critical care medicine.     

The biological distributions of some technetium-MDP components isolated by anion exchange high performance liquid chromatography

Individual components of 99mTc(NaBH4)-MDP mixtures, separated by anion exchange HPLC, have been evaluated as skeletal imaging agents in rats. Biodistribution data show that the evaluated components exhibit markedly different bone uptakes and soft tissue localizations. The component with the highest bone uptake and the highest bone/muscle and bone/blood ratios, is the major component of carrier-added 99mTc(NaBH4)-MDP formulations prepared at pH 8.2. Comparative scintiphotos illustrate the enhanced imaging performance of this component vs unseparated reaction mixtures.     

Characterization of monoclonal antibodies against Schistosoma mansoni

Monoclonal antibodies directed against Schistosoma mansoni antigens were produced by the in vitro fusion of B lymphocytes, obtained from mice infected with S. mansoni, and SP2/0 myeloma cells. Antibody reactivity was assessed by ELISA binding, utilizing 4 M KCl extracts of cercariae and adult worms, soluble egg antigen (SEA), and purified antigenic preparations, and by indirect immunofluorescence using living schistosomula. The monoclonal antibodies recognized a wide spectrum of antigenic determinants. The specificity of the monoclonal reactivities ranged from high cross-reactivity to extreme restriction, vis-a-vis the distribution of the recognized determinants within genus, species, stages, and purified antigenic preparations. The specificity of reactivity of monoclonal antibodies for a given determinant was greater than that of immune mouse serum. These studies establish the feasibility of the production of large numbers of monoclonal antibodies and of their use of antigen identification. The monoclonal antibodies are available to interested investigators upon request.     

The use of high-performance size exclusion chromatography (HPSEC) as a molecular weight screening technique for polygalacturonic acid for use in pharmaceutical applications

Polygalacturonic acid is a linear carbohydrate polymer of monomeric galacturonic acid. It is commercially available as apple and citrus pectins comprised of a mixture of partially methoxylated and/or amidated polygalacturonic acids with molecular weights ranging from 25 000 to >100 000 Da. Pectin can be chemically or enzymatically hydrolyzed to yield polygalacturonic acid fractions of diverse average molecular weight ranges and polydispersities for a variety of uses. Pectin and polygalacturonic acid are used extensively as gelling agents and stabilizers by the food industry, and have applications as therapeutic, and diagnostic pharmaceutical agents such as the magnetic resonance imaging agent LumenHance®. A simple high-performance size exclusion chromatography (HPSEC) method, employing commonly available non-specialized HPLC instrumentation, is described for use as a rapid molecular weight screening technique to determine the average molecular weight range and polydispersity of polygalacturonic acid intended for use in pharmaceutical formulations. A TosoHaas G3000PWXL HPLC column, 50 mM phosphate buffer (pH≈6.9) mobile phase, and refractive index detection were used. A molecular weight calibration curve was linear for polysaccharide standards of 180–100 000 Da with a coefficient of correlation of 0.999. The method was employed to screen commercially available polygalacturonic acid raw materials for average molecular weight data (M_n, M_w, and M_p) and polydispersity (M_w/M_n).     

Delayed type hypersensitivity granuloma formation around Schistosoma mansoni eggs in vitro. IV. Granuloma formation in human schistosomiasis

Peripheral blood mononuclear cells, obtained from 42 school-age Egyptian children, were isolated on Ficoll-hypaque density gradients and assayed for granuloma formation. A granuloma index (G.I.) which classified the cellular reactions to Schistosoma mansoni eggs was determined for each patient. Morphologic criteria to assess the cellular reactivity included cell adherence, blast cell transformation, cell migration, and circumoval accumulation of inflammatory cells around the egg. The difference between the mean granuloma index of uninfected controls (G.I. = 1.25 +/- 0.04) and infected patients (G.I. = 1.58 +/- 0.05) was statistically significant (P less than 0.01; Student's t-test). There was no correlation between the granuloma indices and infection intensities determined by quantitative egg counts or between anti-major serological antigen antibody titers. These data demonstrate the feasibility of studying granulomatous hypersensitivity in human schistosomiasis utilizing an in vitro model of granuloma formation and peripheral blood cells.