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Biomedical subjects

M E Wiebe

Publications and source records attributed to M E Wiebe.

18 recordsLinked to original sources
PubMed1990

Cell banking.

The use of cultured cells (diploid strains or continuous cell lines) to produce biopharmaceuticals provides a level of standardization to the manufacturing process that cannot be attained by the more traditional methods of biological extraction from animal or human fluids, tissues, or primary cells. The key to this advantage is the ability to cryopreserve the production cell line as a master cell bank. This bank serves as the common starting source of a given product for the lifetime of the manufacture of that product. Since the MCB is the common and only starting source, it can be exhaustively characterized with regard to contamination by adventitious and endogenous agents. Assays can be developed for cellular components that are potential contaminants of the product. The removal of these components during product purification can be validated and their removal confirmed by rigorous quality control. In conclusion, characterized cell banks are central to the standardization of biopharmaceutical manufacturing processes and give rise to the production of high-quality products not attainable by the traditional methods of extraction of product from sources which are continually changing.

Animals↗
PubMed1990

Process validation for cell culture-derived pharmaceutical proteins.

Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.

Animals↗
PubMed1987

Effect of immunosuppressants on OKT3 associated T cell activation: clinical implications.

The monoclonal antibody directed at the T cell differentiation antigen T3 (CD3, T.gp 20-25) appears to be superior to conventional high-dose steroids in the treatment of rejection in cadaveric renal graft recipients. Re-rejection episodes and other adverse reactions, probably secondary to T cell activating potential of anti-T3, continue to be clinical problems with anti-T3 therapy. We therefore examined the relative efficacy of cyclosporin A (CSA), methylprednisolone (MP), or 6-mercaptopurine (6-MP), at concentrations that are readily accomplished in clinical practice, on the activation of T cells by anti-T3. CSA or MP mediated marked and 6-MP mediated modest inhibition of anti-T3 induced proliferation of alloimmune memory T cells. CSA- or MP-inhibited anti-T3 elicited specific secondary cytolytic activity and natural killer (NK) cell activity, and 6-MP failed to prevent the augmentation of NK cell activity mediated by anti-T3. The immunosuppressants also exhibited differential effects on anti-T3-associated lymphokine production by peripheral blood mononuclear cells. Interleukin 2 production was completely inhibited by CSA, modestly inhibited by MP and not inhibited by 6-MP. Interferon gamma production was completely inhibited by CSA or MP and not inhibited by 6-MP. Our findings, in addition to providing a plausible immunological basis for some of the complications of anti-T3 therapy, provide experimental support for therapeutic strategies that include the use of CSA and/or MP along with anti-T3.

Antibodies, Monoclonal↗
PubMed1985

Markers of Venezuelan encephalitis virus which distinguish enzootic strains of subtype I-D from those of I-E.

Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.

Animals↗
PubMed1984

Activation of human macrophages. Comparison of other cytokines with interferon-gamma.

Cytokines affecting mononuclear phagocytes were screened for activation of human macrophages to secrete H2O2 and kill toxoplasmas. In contrast to recombinant interferon-gamma (rIFN gamma), the following factors, tested in partially or highly purified form and over a wide range of concentrations, did not augment these functions: native interferon-alpha (nIFN alpha), rIFN alpha A, rIFN alpha D, rIFN beta, colony stimulating factor (type 1) (CSF-1), CSF for granulocytes and macrophages (GM-CSF), pluripotent CSF (p-CSF), tumor necrosis factor (TNF), native interleukin 2 (nIL-2), and rIL-2. Partially purified migration inhibitory factor (MIF) enhanced H2O2-releasing capacity submaximally without inducing antitoxoplasma activity, and warrants further study.

Biological Products↗
PubMed1984

Specificity of a monoclonal antibody for the NH2-terminal region of fibrin.

A monoclonal antibody (MAb/T2G1s) was prepared by fusion using spleen cells from mice immunized with the NH2-terminal CNBr fragment of human fibrin II, the so-called (T)N-DSK [(A alpha 17-51, B beta 15-118, gamma 1-78)2]. In competition experiments, this antibody reacted with (T)N-DSK as well as peptide B beta 15-42 which can be obtained from (T)N-DSK by digestion with plasmin. Little or no reaction was observed with intact fibrinogen, the NH2-terminal CNBr fragments from fibrinogen (N-DSK) or fibrin I [(B)N-DSK], respectively, as well as peptide B beta 1-42. These results suggest that MAb/T2G1s is directed to an epitope on the B beta chain in fibrin II but not in fibrinogen or fibrin I. As such, MAb/T2G1s differs completely from another antibody (MAb/1-8C6)--also specific for the NH2-terminal region of the B beta chain--which was recently described [Kudryk et al. (1983) Molec. Immun. 20, 1191-1200].

Animals↗
PubMed1983

Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity.

Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of 1 X 10(6) and 10(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of 19.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- 1.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.1 antiviral U/ml of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.

Adult↗
PubMed1983

A monoclonal antibody with ability to distinguish between NH2-terminal fragments derived from fibrinogen and fibrin.

A stable hybridoma secreting homogeneous antibody (immunoglobulin class IgG2a) has been prepared by fusion using cells of immunoglobulin non-secreter myeloma (P3X63Ag8.653) and spleen cells of mice which had previously been immunized with the NH2-terminal CNBr fragment of human fibrinogen, the so-called N-DSK [(A alpha 1-51, B beta 1-118, gamma 1-78)2]. In competitive ELISA or radioimmunoassay this antibody (MAb/1-8C6) cross-reacted with intact fibrinogen, N-DSK, a des fibrinopeptide A (des FPA) variant of N-DSK, the so-called (B)N-DSK, as well as the intact B beta chain (B beta 1-118) obtained from N-DSK. Also, and mot importantly, cross-reactivity was observed with fibrinogen-free ethanol extracts of plasma obtained from patients known to contain high levels of fibrinogen or fibrin degradation products. In vitro thrombin digestion of any of these competitors resulted in complete loss of cross-reactivity. MAb/1-8C6 did not react with the A alpha or gamma-chains of N-DSK, free fibrinopeptide B(FPB), free B beta 15-42, as well as equimolar mixtures of the latter two peptides. These results suggest that MAb/1-8C6 may be to an epitope in or around the thrombin-susceptible B beta 14 Arg-25 Gly bond. Furthermore, due to its reactivity with patient plasma extracts, this antibody may be useful in clinical investigations dealing with fibrino(geno)lysis.

Antibodies, Monoclonal↗
PubMed1983

Marker characteristics of Venezuelan encephalitis virus strains isolated before and after epidemics and equine epizootics in Middle America.

Ninety-four strains of Venezuelan encephalitis (VE) virus isolated from sentinel hamsters exposed in the Middle American countries of Mexico, Guatemala, Belize, and Honduras were examined for the presence of virions with marker characteristics of strains that cause large epidemics and equine epizootics. Thirty-four strains came from before and 60 strains came from after the Middle American epidemics and equine epizootics of 1966 and 1969-1972. Twenty-three virion clones that resembled epizootic strains by hydroxylapatite chromatography and Vero monkey kidney cell plaque size determinations were characterized further. However, the predominant virions in these clones were like enzootic strains from Middle America north of the Panama Canal region, and not like Middle American epizootic VE strains, since they were in hemagglutination-inhibition antigenic subtype IE, usually had optimal pH of hemagglutination at 6.2, and were avirulent for English shorthair guinea pigs inoculated subcutaneously. These results provide evidence against the theory of origin of epidemic-equine epizootic VE virus strains that posits that epizootic virions emerge in Middle America from strains containing mixtures of enzootic and epizootic virions in enzootic habitats.

Animals↗
PubMed1979

A hamster-attenuated, temperature-sensitive mutant of Venezuelan encephalitis virus.

Pathogenicities of 10 temperature-sensitive mutants of Venezuelan encephalitis virus were studied using the hamster model of human virulence. The parental strain and nine of the temperature-sensitive mutants produced lethal infections in hamsters. Strain ts 126 showed reduced hamster virulence. Deaths with the lethal mutants usually occurred 1 to 3 days later than with parental virus. Nine mutants produced lower levels of viremia than parental virus. Attenuation of ts 126 was related to restriction of viral growth in spleen and probably bone marrow and to absence of the usual pathological lesions in hemopoietic tissues and brain, but was functionally unrelated to temperature sensitivity since temperatures of both normal and infected hamsters remained within the permissive range of the mutant. Deaths did not correlate with titers of the 10 mutants in blood at permissive temperatures or with reversions of four temperature-sensitive mutants to non-temperature-sensitive virus in hamsters.

Animals↗
PubMed1972

Isolation and characterization of two cell types of Coxiella burneti phase I.

Two morphologically distinct cell types of Coxiella burneti phase I have been separated on the basis of unique buoyant densities. When centrifuged to equilibrium in cesium chloride or density gradients of sucrose or Renografin, the cells band in two zones. Electron micrographs of ultrathin sections of the two cesium chloride-separated cell types indicate a considerable number of morphological differences. The lower-density cells are small, compact, and rodshaped and have very dense nucleoids. The cell type of highest density is larger, rounded, and more pleomorphic, and the nucleoid filaments are more dispersed. The two cell types are nearly identical in sedimentation rates, and both infect chick yolk sac cells and are lethal to chick embryos. They convert to a mixture of cell types when cultured separately. Treatment with Formalin induces all cells to band at the same position when centrifuged to equilibrium in cesium chloride. The cell type variance was found to be independent of the antigenic phase phenomenon of C. burneti.

Animals↗
PubMedDate not supplied

Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations.

Use of the organic solvent, tri(n-butyl)phosphate (TNBP), and detergents for the inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation obtained with TNBP plus Tween 80 was superior to that observed with ethyl ether plus Tween 80, a condition previously shown to inactivate greater than or equal to 10(6.9) CID50 of hepatitis B and greater than or equal to 10(4) CID50 of Hutchinson strain non-A, non-B hepatitis. The AHF recovery after TNBP/Tween treatment was greater than or equal to 90 percent. Following the reaction, TNBP could be removed from the protein by gel exclusion chromatography on Sephadex G25; however, because of its large micelle size, Tween 80 could not be removed from protein by this method. Attempts to remove Tween 80 by differential precipitation of protein were only partially successful. An alternate detergent, sodium cholate, when combined with TNBP, resulted in almost as efficient virus inactivation and an 80 percent recovery of AHF. Because sodium cholate forms small micelles, it could be removed by Sephadex G25 chromatography. Electrophoretic examination of TNBP/cholate-treated AHF concentrates revealed few, if any, changes in protein mobility, except for plasma lipoprotein(s).

Blood Proteins↗
PubMedDate not supplied

Inactivation of viruses in labile blood derivatives. II. Physical methods.

The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure to lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated.

Citrates↗