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Conjugates of nonimmunogenic valency platform molecules and analogs of immunogens that possess the specific B cell binding ability of the immunogen but lack T cell epitopes and which, when introduced into individuals, induce humoral anergy to the immunogen are disclosed. Accordingly, these conjugates are useful for treating antibody-mediated pathologies that are caused by foreign or self immunogens.
1. A method of identifying a polypeptide analog of a T cell-dependent immunogen implicated in an antibody-mediated pathology comprising the steps of:
(a) screening a panel or library of candidate polypeptides for the presence of one or more B cell epitope(s) that specifically binds to antibodies to which the immunogen binds specifically;
(b) screening said candidate polypeptides identified in step (a) for candidate polypeptides lacking T cell epitope(s); and
(c) selecting a polypeptide analog from said candidate polypeptides that bind to the antibodies of step (a), but that do not contain T cell epitope(s) as determined by step (b).
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6. A method of making a polypeptide analog of a T cell-dependent immunogen implicated in an antibody-mediated pathology comprising the step of removing T cell epitope(s) from a polypeptide comprising an epitope that binds specifically to antibodies to which the immunogen binds specifically.
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13. A method of making a polypeptide analog of a T cell-dependent immunogen implicated in an antibody-mediated pathology comprising the steps of:
(a) synthesizing the polypeptide analog, whereby the polypeptide analog comprises an epitope that binds specifically to antibodies to which the immunogen binds specifically, wherein the analog lacks T cell epitopes capable of activating T cells in an individual; and
(b) purifying the analog.
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This application is a divisional of Ser. No. 10/081,076, filed Feb. 20, 2002, which is a continuation of Ser. No. 09/563,167, filed May 2, 2000, now abandoned, which is a continuation of Ser. No. 08/118,055, filed Sep. 8, 1993, now U.S. Pat. No. 6,060,056, which is a continuation-in-part of Ser. No. 07/652,648, filed Feb. 8, 1991, now U.S. Pat. No. 5,268,454, the disclosures of which are incorporated herein by reference.
This invention is in the field of immunology and concerns compositions and methods for inducing humoral anergy for the purpose of treating antibody-mediated pathologies. More specifically, the invention relates to conjugates of nonimmunogenic valency platform molecules and analogs of immunogens that lack T cell epitopes.
In order to survive in a world of pathogenic or potentially pathogenic microorganisms, higher organisms have evolved immune systems which can specifically recognize virtually any foreign substance through its characteristic molecules. This recognition frequently results in the production of specific proteins called antibodies which bind only to the foreign substance which induced their synthesis, causing the elimination of the invading microorganism. Occasionally an animal's immune system makes antibodies which recognize some of its own molecules, generating an autoimmune state that may affect the animal's health adversely.
The induction of specific antibodies in response to an immunogen involves the interaction of multiple cell types, including thymus-derived lymphocytes (T cells), macrophages, and bone marrow-derived lymphocytes (B cells). This is in contrast to the primary (IgM) immune response which does not include T Cells. T cell dependent antigen responses are secondary responses. B cells possess surface immunoglobulin by which they are able to bind immunogens, the first step in their activation and clonal expansion. A single B cell expresses only one type of antigen-specific immunoglobulin. The site(s), region(s) or domain(s) of the immunogen to which the immunoglobulin binds is called a "B cell epitope." In the second step of B cell activation and expansion, T cells are activated through interaction with a site, region or domain of the immunogen called a "T cell epitope" which is presented by B cells or other antigen-presenting cells. Once activated, the T cells provide positive signal(s) to the B cells to which the immunogen is bound and they proceed to differentiate and to produce and secrete antibody. Positive signals from the T cell include the secretion of lymphokines, and/or direct contact between the B cells and T cells. T cell epitopes may be different or more restricted in scope than B cell epitopes. As discussed above, in order for an immunogen to elicit T dependent antibodies, it must have epitopes recognized by both B and T cells.
Past attempts to treat antibody-mediated pathologies have involved both general and specific suppression of the immune response. General suppression has typically employed broad spectrum, nonspecific immunosuppressants such as cyclophosphamide or steroids. Because these nonspecific drugs suppress many aspects of the immune system, they limit its required and beneficial functions as well as the malfunction causing the condition being treated. They are thus used only with extreme caution and subject the patient to risk from secondary infections or other undesirable side effects.
Because of the disadvantages of general immunosuppression, methods for specifically suppressing an immune response to an immunogen without affecting the normal functions of the immune system are highly preferred for treating antibody-mediated pathologies. The present invention concerns compositions and methods for specifically suppressing the humoral response to immunogens.
Prior attempts to induce specific immunosuppression have focused on conjugating haptens and immunogens to nonimmunogenic polymeric carriers. Benacerraf, Katz and their colleagues used conjugates of haptens and antigens and copolymers of D-lysine and D-glutamic acid (formerly D-GL, hereinafter D-EK). Their initial studies involved conjugates of the synthetic hapten 2,4-dinitrophenyl (DNP) in guinea pigs and mice and showed the conjugates were capable of inducing humoral unresponsiveness. These initial studies were then extended to conjugates of other haptens and conjugates of immunogens. While the results with haptens were repeatable, and although their patents (U.S. Pat. Nos. 4,191,668 and 4,220,565) allege the approach is effective in inducing tolerance to immunogens, subsequent work has shown that conjugates of D-EK and immunogens do not provide a means for inducing humoral unresponsiveness to the immunogen. For instance, Liu et al., J. Immun. (1979) 123:2456–2464, report that subsequent studies of those conjugates demonstrate that the conjugates "do not induce unresponsiveness at the level of protein specific B cells." Similarly, Butterfield et al., J. Allergy Clin. Immun. (1981) 67:272–278, reported that conjugates of ragweed immunogen and D-EK actually stimulated both IgE and IgG responses to the immunogen.
This subsequent work and other data dealing with conjugates of nonimmunogenic polymers and immunogens (Saski et al., Scand. J. Immun. (1982) 16:191–200; Sehon, Prog. Allergy (1982) 32:161–202; Wilkinson et al., J. Immunol. (987) 139:326–331, and Borel et al., J. Immunol. Methods (1990) 126:159–168) appear to indicate that the anergy, if any, obtained with such conjugates is due to suppression by T cells to directly suppress the immune response.
Several other references deal with conjugates of nonimmunogenic polymers and DNA. See U.S. Pat. Nos. 4,191,668; 4,650,625; J. Clin. Invest. (1988) 82:1901–1907; and commonly owned U.S. patent application Ser. No. 07/494,118. As a whole, these references indicate that these DNA conjugates may suppress the production of antibodies to this lupus autoimmunogen. It should be noted in this regard that DNA is not immunogenic and does not possess T cell epitopes.
In sum, applicants believe the prior art shows that antibody production to conjugates of nonimmunogenic stable polymers and haptens or DNA, neither of which have T cell epitopes, may provide B cell unresponsiveness. Applicants also believe that conjugates of immunogens do not provide B cell unresponsiveness but may activate T cells to directly suppress the immune response.
The present invention resides in the discovery that the failure of the prior conjugates of nonimmunogenic polymers and immunogens to induce B cell anergy (unresponsiveness) was due to the fact that the immunogens contained both B and T cell epitopes and that if the latter were eliminated, the conjugate would be effective for inducing B cell anergy.
Accordingly, one aspect of the invention is a composition for inducing specific B cell anergy to an immunogen comprising a conjugate of a nonimmunogenic valency platform molecule and an analog of the immunogen that (a) binds specifically to B cells to which the immunogen binds and (b) lacks the T cell epitope(s) of the immunogen.
Pharmaceutical compositions of the above-described conjugates and pharmaceutically acceptable carriers or vehicles are another aspect of the invention.
A further aspect of the invention is a method of inducing specific B cell anergy to an immunogen in an individual comprising administering to the individual an effective amount of the above-described conjugate.
Yet another aspect of the invention is a method of treating an individual for an antibody-mediated pathology in which undesired antibodies are produced in response to an immunogen comprising administering to the individual a therapeutically effective amount of the above-described conjugate.
As used herein the term "B cell anergy" intends unresponsiveness of those B cells requiring T cell help to produce and secrete antibody and includes, without limitation, clonal deletion of immature and/or mature B cells and/or the inability of B cells to produce antibody. "Unresponsiveness" means a therapeutically effective reduction in the humoral response to an immunogen. Quantitatively the reduction (as measured by reduction in antibody production) is at least 50%, preferably at least 75%, and most preferably 100%.
"Antibody" means those antibodies which are T cell dependent.
As used herein the term "immunogen" means a chemical entity that elicits a humoral immune response when injected into an animal. Immunogens have both B cell epitopes and T cell epitopes.
The term "analog" of an immunogen intends a molecule that (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes. Although the analog will normally be a fragment or derivative of the immunogen and thus be of the same chemical class as the immunogen (e.g., the immunogen is a polypeptide and the analog is a polypeptide), chemical similarity is not essential. Accordingly, the analog may be of a different chemical class than the immunogen (e.g., the immunogen is a carbohydrate and the analog is a polypeptide) as long as it has the functional characteristics (a) and (b) above. The analog may be a peptide, carbohydrate, lipid, lipopolysaccharide, nucleic acid or other biochemical entity. Further, the chemical structure of neither the immunogen nor the analog need be defined for the purposes of this invention.
An analog of an immunogen may also comprise a "mimotope." The term "mimotope" intends a synthetic molecule which competitively inhibits the antibody from binding the immunogen. Because it specifically binds the antibody, the mimotope is considered to mimic the antigenic determinants of the immunogen. Like an analog of an immunogen, a mimotope (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes.
An analog of an immunogen may also comprise an "aptamer." The term "aptamer" intends a synthetic oligonucleotide which competitively inhibits the antibody from binding the immunogen. Like an analog of an immunogen, an aptamer (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes.
As used herein "valency platform molecule" means a nonimmunogenic molecule containing sites which facilitate the attachment of a discreet number of analogs of immunogens.
"Nonimmunogenic" is used to describe the valency platform molecule and means that the valency platform molecule elicits substantially no immune response when it is administered by itself to an individual.
As used herein "individual" denotes a member of the mammalian species and includes humans, primates, mice and domestic animals such as cattle and sheep, sports animals such as horses, and pets such as dogs and cats.
Immunogens that are involved in antibody-mediated pathologies may be external (foreign to the individual) immunogens such as allergens, Rh hemolytic disease (D immunogen), biological drugs, including native biological substances foreign to the individual such as therapeutic proteins, peptides and antibodies, and the like or self-immunogens (autoimmunogens) such as those associated with thyroiditis (thyroglobulin), stroke (cardiolipin), male infertility (α-sperm), myasthenia gravis (acetylcholine receptor) and rheumatic-fever (carbohydrate complex).
Analogs to such immunogens may be identified by screening candidate molecules to determine whether they (a) bind specifically to serum antibodies to the immunogen and (b) lack T cell epitopes. Specific binding to serum antibodies may be determined using conventional immunoassays and the presence or absence of T cell epitopes may be determined by conventional T cell activation assays. In this regard an analog which "binds specifically" to serum antibodies to the immunogen exhibits a reasonable affinity thereto. The presence or absence of T cell epitopes may be determined using the tritiated thymidine incorporation assay described in the examples. The presence of T cell eptiopes can also be determined by measuring secretion of T cell-derived lymphokines by methods well known in the art. Analogs that fail to induce statistically significant incorporation of thymidine above background are deemed to lack T cell epitopes. It will be appreciated that the quantitative amount of thymidine incorporation may vary with the immunogen. Typically a stimulation index below about 2–3, more usually about 1–2, is indicative of a lack of T cell epitopes.
A normal first step in identifying useful analogs is to prepare a panel or library of candidates to screen. For instance, in the case of protein or peptide analogs, libraries may be made by synthetic or recombinant techniques such as those described by Geysen et al. in Synthetic Peptides as Antigens; Ciba Symposium (1986) 119:131–149; Devlin et al., Science (1990) 249:404–406; Scott et al., Science (1990) 249:386–390; and Cwirla et al., PNAS USA (1990) 87:6378–6382. In one synthetic technique, peptides of about 5 to 30 amino acids are synthesized in such a manner that each peptide overlaps the next and all linear epitopes are represented. This is accomplished by overlapping both the carboxyl and amino termini by one less residue than that expected for a B cell epitope. For example, if the assumed minimum requirement for a B cell epitope is six amino acids, then each peptide must overlap the neighboring peptides by five amino acids. In this embodiment, each peptide is then screened against antisera produced against the native immunogen, either by immunization of animals or from patients, to identify the presence of B cell epitopes. Those molecules with antibody binding activity are then screened for the presence of T cell epitopes as described in the examples. The molecules lacking T cell epitopes are useful as analogs in the invention.
If the T cell epitope(s) of an immunogen are known or can be identified, random T cell screening of candidate analogs is not necessary. In such instances, the T cell epitope(s) may be altered (e.g., by chemical derivatization, or elimination of one or more components of the epitope) to render them inoperative or be eliminated completely, such as, for instance, in the case of peptides, by synthetic or recombinant procedures.
Mimotopes and aptamers are synthesized by conventional methods and are screened in the same manner as other analogs of immunogens.
The analogs are coupled to a nonimmunogenic valency platform molecule to prepare the conjugates of the invention. Preferred valency platform molecules are biologically stabilized, i.e., they exhibit an in vivo excretion half-life often of hours to days to months to confer therapeutic efficacy, and are preferably composed of a synthetic single chain of defined composition. They will normally have a molecular weight in the range of about 200 to about 200,000, usually about 200 to about 20,000. Examples of valency platform molecules within the present invention are polymers such as PEG, poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, immunoglobulins, and D-EK. Preferred polymers are based on polyethylene glycols (PEGs) having a molecular weight of about 200 to about 8,000. Other preferred polymers are D-EKs having a molecular weight of about 5,000 to about 30,000, and an E:K (D-glutamic acid:D-lysine) mole ratio of approximately 60:40, as described in U.S. patent application Ser. No. 07/494,118; referenced above.
Conjugation of the analog to the valency platform molecule may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the analog and valency platform molecule.
Polypeptide analogs will contain amino acid sidechain groups such as amino, carbonyl, or sulfhydryl groups that will serve as sites for coupling the analog to the carrier. Residues that have such functional groups may be added to the analog if the analog does not already contain same. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. In the case of carbohydrate or lipid analogs, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent. In a similar fashion the valency platform molecule may also be derivatized to contain functional groups if it does not already possess appropriate functional groups. With specific reference to conjugating peptide analogs and D-EK or other proteinaceous valency platform molecules, coupling is preferably carried out using a heterobifunctional crosslinker, such as sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, which links the ε amino group on the D-lysine residues of D-EK to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled. This method is usually carried out such that an average of 3 to 5 analog molecules are coupled to each D-EK molecule and the average molecular weight of the D-EK prior to coupling is 5,000 to 30,000 daltons.
The conjugates will normally be formulated for administration by injection (e.g., intraperitoneally, intramuscularly, etc.). Accordingly, they will typically be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. The conjugate will normally constitute about 0.01% to 10% by weight of the formulation. The conjugate is administered to an individual in a "therapeutically effective amount", i.e., an amount sufficient to produce B cell anergy to the involved immunogen and effect prophylaxis, improvement or elimination of the antibody-mediated condition being addressed. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Normally, a dose of about 10 μg to 1 mg conjugate/kg body weight will be given, daily for three consecutive days. Other appropriate dosing schedules would be 3 doses per week, or one dose per week, or one dose every two to four weeks, or one dose on a monthly or less frequent schedule depending on the individual or the disease state. Repetitive administrations, normally timed according to B cell turnover rates may be required to achieve and/or maintain a state of humoral anergy. Such repetitive administrations will typically involve treatments of up to 1 mg/kg of body weight every 30 to 60 days, or sooner, if an increase in antibody titer is detected. Alternatively, sustained continuous release formulations of the conjugates may be indicated for some pathologies. Various formulations and devices for achieving sustained release are known in the art.
Anti-T helper cell treatments may be administered together with the conjugates. Such treatments usually employ agents that suppress T cells such as steroids or cyclosporin.
The following examples are intended to further illustrate the invention and its uniqueness. These examples are not intended to limit the scope of the invention in any manner.
Preparation of Peptides and D-EK/Peptide Conjugates:
The α-subunit of the acetylcholine receptor of Torpedo californicus is described by Stroud, R. M., and Finer-Moore, J., Ann. Rev. Cell Biol. (1985) 1:317:351, and Sumikawa, K., et al., Nucl. Acids Res. (1982) 10:5809–22. The peptide defined by residues 47–127 of that α-subunit is called the major immunogenic region (MIR).
Two peptides, L-42 and L-53, corresponding to residues 61–77 and 112–127 of that α-subunit, were synthesized using conventional solid-phase methods and purified to homogeneity by HPLC. An amino terminal cysteine was added to each sequence for the purpose of attachment of the peptide to D-EK via a thio ether linkage.
Each peptide (40 mg) was dissolved in 0.1 M sodium borate buffer, pH 9.0. The solution was reacted with citraconic anhydride (400 μL) at room temperature; the pH was maintained above 7.0 by addition of 1 M NaOH. The solution was then made 20 mM in dithiothreitol and was warmed at 37° C. for 20 minutes to reduce the peptide. The mixture was quickly desalted over G-10 Sephadex columns which were equilibrated with 0.1M sodium borate, pH 7.0.
D-EK (200 mg, weight average molecular weight ≃10,000–30,000) was dissolved in 2.0 mL of 0.1M sodium borate. Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (SSIAB, 10 mg, Pierce Chemical) was added to the mixture and the mixture was reacted for 90 minutes at room temperature in the dark. The mixture was then desalted over a 10 mL G-25 column, equilibrated with 0.1M sodium borate, pH 7.0.
The desalted SSIAB-D-EK was mixed with the reduced and desalted peptide and reacted overnight. The resulting conjugate was placed in dialysis tubing with a 14 Kd cutoff and was dialyzed against 5% acetic acid to remove citraconyl groups. The dialysis buffer was changed to phosphate-buffered saline and the dialysis continued.
Detection of B Cell Epitopes:
CAF1 mice were obtained and housed at the La Jolla Pharmaceutical animal facility according to National Institutes of Health guidelines. CAF1 mice were immunized (day 0) intraperitoneally (i.p.) with 50 μg of recombinant torpedo MIR absorbed onto alum plus B. pertussis vaccine (B. pertussis vaccine obtained from Michigan Department of Public Health, Lansing, Mich.) (Iverson, G. M., (1986) Handbook of Experimental Immunology, Vol.2, p. 67, D. M Weir ed., Blackwell Scientific Publications, Palo Alto, Calif.). The mice received a booster injection of the same protein in saline, i.p., on day 21 and were bled from the tail vein on day 28. Sera from these mice (anti-MIR sera) were used to screen peptides L-42 and L-53 for the presence of B cell epitopes, as follows. The sera were added to the wells of microtitration plates which were coated with 10 μg/mL of the indicated peptide conjugates. The plates were incubated at 37° C. for one hour, washed 3 times, 100 μl of alkaline phosphatase-conjugated goat anti-mouse antibody was added, incubated at 37° C. for one hour, washed 3 times, and 100 μl of developer (substrate) was added to each well. The plates were incubated at room temperature for 30 minutes and the amount of color in each well was determined in a Titertek® Multiskan microplate reader. Results are illustrated graphically in
Detection of T Cell Epitopes:
T cell activation was assayed by the general procedure of Bradley, M. L., (1980) in Mishell and Shigii, eds., Selected Methods in Cellular Immunology (W.H. Freeman and Co., San Francisco, Calif.), p. 164. CAF1 mice were obtained and housed at the La Jolla Pharmaceutical animal facility according to National Institutes of Health guidelines. CAF1 mice were immunized in the footpad with 50 μg MIR in Complete Freund's Adjuvant (CFA) on day 0. On day 7 the popliteal lymph nodes were removed and placed in culture in microtiter plates using 5×105 cells per well. The peptides or peptide-DEK conjugate were added to the cultures, and on day 4, 1 μCi of tritiated thymidine was added to each well to measure proliferation of T cells. The cultures were harvested on day 5 with a Skatron® cell harvester. The amount of incorporated 3H-thymidine was determined in a Beckman L6800® liquid scintillation counter. The stimulation index was calculated by dividing the CPM incorporated with peptide by the CPM incorporated from cultures without any peptide. A stimulation index >2–3 was indicative of the presence of a T cell epitope on the peptide added to the well. As shown in
Induction of B Cell Anergy to L-53 by L-53-D-EK Conjugate:
CAF1 mice were obtained and housed at the La Jolla Pharmaceutical animal facility according to National Institutes of Health guidelines. CAF1 mice were immunized with 50 μg of MIR, i.p., absorbed onto alum plus B. pertussis vaccine on day 0. On days 21, 22 and 23 the mice (6 mice per group) received 10 or 100 μg of either L-42-D-EK conjugate or L-53-D-EK conjugate. One group received only saline. On day 28 all mice received a booster injection of MIR in saline and on day 35 all mice were bled and assayed for the presence of antibodies to L-42 and L-53 in their sera, using an ELISA assay as described above with respect to
This example is further evidence that conjugates of immunogens and D-EK do not induce B cell anergy.
Synthesis of Ovalbumin-D-EK Conjugate:
Chicken egg ovalbumin (ova; 50 mg) was dissolved in 5 mL of 0.1M sodium borate buffer, pH 9.0, containing 10 mM EDTA. After the addition of 3.0 mg of 2-iminothiolane (Traut's reagent), the mixture was reacted for 2.5 hours at room temperature. D-EK (54 mg), dissolved in 0.5 M sodium borate, pH 9.0, at a concentration of 100 mg/mL, was reacted with SSIAB (18 mg; Pierce Chemical) for 2.5 hours in the dark, at room temperature. The two reaction mixtures described above were desalted separately on G-25 columns (Pharmacia; 10 mL column volume, equilibrated with 0.1 M sodium borate, pH 9.0) and the excluded fractions were combined and reacted for 16 hours at 4° C., in the dark. The reaction product was fractionated by gel filtration over Sephacryl S-200 (490 mL, Pharmacia) columns, equilibrated with 0.2 M ammonium bicarbonate. Fractions containing conjugate, as assessed by polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulfate (SDS-PAGE), were pooled and dried under vacuum. The dried material was reacted with 0.8 mL of citraconic anhydride, maintaining the pH between 7 and 9 by the addition of 1M NaOH, in order to efficiently separate conjugated ovalbumin from unreacted protein. The citraconylated conjugate was rechromatographed over S-200, and fractions containing high molecular, weight material (>80,000 daltons), as assessed SDS-PAGE; were used for biological studies.
Chicken Ovalbumin, when Conjugated to D-EK, does not include B Cell Anergy in Mice Immunized to Chicken Ovalbumin:
CAF1 mice were obtained and housed at the La Jolla Pharmaceutical animal facility according to National Institutes of Health guidelines. Female CAF1 mice were primed with ova (100 μg/mouse, i.p.) precipitated on alum, with B. pertussis vaccine added as an adjuvant. Sixteen weeks later, the mice were divided into two groups of six mice each. One group (control) was treated with saline, and the second group was injected with a conjugate of ova and D-EK (ova-D-EK; 200 μg/mouse/day, i.p.). The mice were dosed on three successive days. One week after the first dose, the mice in both groups were boosted, i.p., with ova in saline (100 μg/mouse). One week later, the mice were bled from a tail vein. The plasma was harvested and assayed for the amount of anti-ova antibodies by an ELISA assay. As shown in Table 1, the ova-D-EK conjugate did not suppress the anti-ova response.
This example is still further evidence that conjugates of immunogens and D-EK do not induce B cell anergy.
Synthesis of MIR-D-EK Conjugate:
MIR was modified on its carboxyl-terminus to include a sequence of 8-amino acids (Arg-Ser-Lys-Ser-Lys-Ser-Lys-Cys (SEQ. ID NO.: 1)). The amino-terminus was extended by one amino acid, proline. Purified modified MIR (250 mg) was reduced with 100 mM dithiothreitol and was desalted over Sephadex G-25 (Pharmacia), equilibrated with 0.1 M sodium borate buffer, pH 9.0, containing 10 mM EDTA. D-EK (400 mg) was reacted with SSIAB (29 mg) as in the previous examples. The product was desalted over G-25. The excluded volumes from the modified MIR and D-EK G-25 column runs were combined and reacted at 4° C. for 16 hours, in the dark. Excess SSIAB groups were quenched with 2-mercaptoethanol, and the reaction mixture was concentrated to 20 mL over a PM-10 membrane (Amicon Corporation). The mixture was treated with 1.0 mL of citraconic anhydride and chromatographed over S-300 (Pharmacia; 1.8 L), equilibrated with 5% ammonium hydroxide. Fractions containing two or more modified MIR groups per D-EK; as assessed by SDS-PAGE, were pooled and used for biological studies.
MIR-D-ER Conjugate Contains T Cell Epitopes Recognized by Rats Immunized with MIR from the Same Species:
T cell activation was assayed by the general procedure of Bradley, supra. Female Lewis rats were immunized in the footpad with MIR (50 μg) in complete Freund's adjuvant (CFA) on day 0. On day 7, the popliteal lymph nodes were removed and placed in culture in microtiter plates using 5–105 cells per well. MIR-D-EK was added, and, after, four days of culture, the wells were pulsed with tritiated thymidine (1-μCi) to measure proliferation of T cells. The cultures were collected after 5 days of culture with a Skatron™ cell harvester. The amount of incorporated 3H-thymidine was determined by scintillation spectrometry. The stimulation index was calculated by dividing the counts incorporated in the absence of the conjugate. A stimulation index of greater than 2–3 was considered indicative of the presence of a T cell epitope on the added conjugate. The stimulation index was 4 or greater at all concentrations of MIR-D-EK tested (10 μg/mL to 400 mg/mL), proving that T cells from MIR-immunized rats recognize T cell epitopes on the MIR-D-EK conjugate in this assay.
MIR-D-EK does not Induce B Cell Anergy in Rats Immunized with MIR:
Female Lewis rats were primed with MIR (100 μg/rat) in CFA. Six months later, the rats were divided into three groups of three rats each. One group was treated with saline (control) and the other two groups were treated with MIR-D-EK (100 μg/rat, i.p.) on three successive days. After one week, the rats in the control group and one group that had been treated with MIR-D-EK were boosted with recombinant MIR (1000 μg/rat, i.p.) in saline. One week later, all three groups of rats were bled from the tail vein. The plasma was harvested and assayed for the amount of anti-MIR antibodies by an ELISA assay. Table 2 below reports the data from those assays.
As shown in Table 2, the data on Group 1 animals (saline control) indicate that MIR itself is an immunogen. The data for the Group 2 and 3 animals indicate that the MIR-D-EK conjugate did not suppress the anti-MIR response. In fact, MIR-D-EK boosted the anti-MIR response in Group 3.
These tests, taken together with the results of Example 1 show that the moiety conjugated to D-EK will cause anergy in B cells recognizing that moiety if the moiety either does not contain a T cell epitope or is not recognized by T cells.
Synthesis of L-42 Peptide-KLH Conjugate:
Reduced L-42 (see Example 1) was conjugated to keyhole limpet hemocyanin (KLH) using thioether chemistry similar to that described above with respect to D-EK.
L-42 does not Activate T Cells in Mice Immunized with L-42-KLH:
Activation of T cells by peptides was measured by the general procedure of Bradley, supra. Female CAF1 mice were immunized in the footpad with L-42 peptide conjugated KLH (L-42-KLH; 50 μg) in CFA on day 0. On day 7, the popliteal lymph nodes were removed and placed in culture in microtiter plates, at a cell density of 5·105 cells/well. Peptides were added, and, after four days of culture, the wells were pulsed with 1 μCi of tritiated thymidine to measure proliferation of T cells. The cultures were collected after 5 days of culture with a Skatron™ cell harvester. The amount of incorporated 3H-thymidine was determined by scintillation spectrometry. The stimulation index was calculated by dividing the counts incorporated in the absence of peptide. An index of greater than 2–3 is indicative of the presence of a T cell epitope on the added peptide.
The data in
L-42-D-EK Conjugate Induces a B Cell Anergy in Mice Immunized to L-42-KLH:
CAF1 mice were primed with 100 μg/mouse of L-42-KLH on alum plus B. pertussis vaccine as an adjuvant. Three weeks later, the mice were divided into groups of six mice each. One group was treated by i.p. injections on three successive days with saline (control); the other groups were similarly treated with various doses of L-42-D-EK (i.p.). Five days later, all mice were boosted with L-42-KLH (50 μg/mouse), and, after a wait of one week, they were bled from the tail vein. The plasma was harvested and assayed for the amount of anti-L-42 and anti-KLH antibodies by ELISA assays. Data are expressed as a percent of a standard serum. An asterisk indicates that a data point was significantly different from the control as determined by a Student t test.
The data in
The melittin molecule, composed of 26 amino acids, is one of the major components of bee venom. One third of the bee venom sensitive individuals have melittin specific antibodies. Melittin is highly immunogenic in some mouse strains (Balb/c, CAF1). The majority (>80%) of melittin-specific antibodies in the responder mouse strains bind a B cell epitope which is the C-terminal heptapeptide of melittin.
Melittin peptides were synthesized using standard Fmoc chemistry techniques on a glycine resin (Advanced ChemTech #SG5130 or equivalent (Advanced ChemTech, 2500 Seventh Street Road, Louisville, Ky.) using 2.3 M excess amino acid derivatives for each coupling step. Completion of the coupling was monitored with bromphenol blue and confirmed with ninhydrin.
Melittin Peptides Used in Conjugations
A cysteine was added as required for coupling certain peptides via a thioether bond to the valency platform molecule. Peptides were purified by reversed phase HPLC following synthesis and lyophilized to dryness. The appropriate amount of peptide was then weighed out for each conjugation.
Reduction of Preformed Disulfide Bonds:
(Tributylphosphine Method)
All buffers were sparged with helium. The peptide was dissolved in a minimal volume (approximately 10 to 20 mg/mL) of 0.05 M NaHCO3 (pH 8.25). A 1 mL solution of 0.7 M tributylphosphine (TBP; MW=202.32 g/mole; d-0.812 g/mL was prepared by adding 174 μL of TBP to 826 μL of isopropanol (iPrOH). Then, 1:1 equivalents of TBP were added to the peptide solution prepared as described above, mixed well, and allowed to react for 30 minutes to 1 hour with occasional mixing to keep TBP dissolved and/or dispersed in the solution. Complete reduction was confirmed by HPLC.
2.93 g (8.28 mmol, 2.2 eq) of iodoacetic anhydride was added to a stirred suspension of 572 mg (3.76 mmol) of 3,5-diaminobenzoic acid in 19 mL of dioxane at room temperature under N2 atmosphere. The mixture was stirred, covered with foil for 20 hours and partitioned between 50 mL of EtOAc and 50 mL of 1 N HCl solution. The EtOAc layer was washed with brine, dried over MgSO4, filtered, and concentrated on a rotary evaporator to give 3.3 g of tan solid. The material was purified by silica gel chromatography (94/5/1 CH2Cl2/MeOH/HOAc) to yield 992 mg (54%) of compound 1 as a white solid: NMR (DMSO) 3.84 (s, 4H), 7.91 (s, 2H), 8.14 (s, 1H), 10.56 (s, 2H).
117 μL (1.6 mmol, 190 mg) of SOCl2 was added to a solution of 390 mg (0.8 mmol) of 1 in 34 mL of THF. The mixture was refluxed under N2 atmosphere until all solids had dissolved (approximately 30 minutes) to give a clear red-brown solution. The mixture was concentrated on the rotary evaporator and placed under vacuum to provide crude compound 2 as a foamy solid which was used directly in the next step.
570 mg of α, w-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol (0.16 mmol, 3350 g/mol, Sigma) was placed in a tared flask. Toluene (20 mL) was added and water was removed by, azeotropic distillation. The residue was dried under vacuum to give 549 mg of solid and dissolved in 4 mL THF with 89 μL (0.64 mmol) of diisopropylethylamine. The crude acid chloride was dissolved in 4 mL anhydrous THF and added to the mixture over 30 seconds under N2. The mixture was stirred for 16 hours at room temperature and partitioned between 25 mL of 0.1 N HCl and 25 mL of CH2Cl2. The aqueous layer was again extracted with CH2Cl2 and the organic layers were combined, washed with 25 mL of H2O, followed by 50 mL of at NaHCO3 solution. The organic layers were dried with Na2SO4, filtered, and concentrated to give 784 mg of orange oil. Silica gel chromatography (9/1 CH2Cl2/MeOH) yielded 190 mg of colorless oil which was crystallized from hot EtOH/Et2O, collected on sintered glass filter under N2 pressure, and dried under vacuum to provide 177 mg of compound 3 as a white solid: NMR (CDCl3 3.40 (bd m, 8H), 3.59 (bd s, (CH2CH2O)n, integral too large to integrate in relation to other integrals), 3.80 (bd m, 4H), 3.91 (s, 8H), 7.49 (brd m, 2H), 7.77 (bd m, 2H), 7.82 (bd s, 4H), 8.27 (bd s, 2H), 8.90 (bd m, 4H): iodoacetyl determination (European Journal of Biochemistry (1984) 140:63–71): Calculated, 0.92 mmol/g; Found, 0.96 mmol/g.
This compound was prepared as described by Weltman, J. K., 1983 Biotechniques 1:148–152. Briefly, 708 mg (2.0 mmol) of iodoacetic anhydride was added to a solution of 137 mg (1.0 mmol) of para-aminobenzoic acid in 10 mL of dioxane. The mixture was stirred in the dark for 18 hours and partitioned between 25 mL of H2O and 25 mL of EtOAc. The EtOAc layer was washed with saturated NaCl solution, dried (MgSO4), filtered and concentrated to yield 797 mg of a peach colored solid. Recrystallization from hexanes/EtoAc yielded 221 mg (72%) of 4-(iodoacetamido)benzoic acid as a white solid: mp 220–230°; 1H NMR (DMSO) d 3.86 (s, 2H), 7.68 (d, 2H), 7.91 (d, 2H), 10.60 (s, 1H).
188 mg (0.909 mmol) of dicyclohexylcarbodiimide was added to a solution of 185 mg (0.606 mmol) of 4-(iodoacetamido)benzoic acid and 406 mg (0.121 mmol) of α, w-bis-(N-2-aminoethylcarbamolyl)polyethyleneglycol (Sigma Chemical Co., St. Louis, Mo., dried by azeotropic distillation with toluene) in 2 mL of THF. The mixture was stirred for 2 hours and then six drops of acetic acid were added. 10 mL of CH2Cl2 was added and the mixture was kept in a freezer for 30 minutes. The mixture was filtered to remove solids and the filtrate was concentrated to viscous residue. Purification by silica gel chromatography (gradient 99/1 to 96/4 CH2Cl2/MeOH) provided a solid which was triturated with MeOH to give 292 mg of a cream colored solid: 1H (CDCl3) 3.48 (m, 8H), 3.63 (bd s, (CH2CH2O)n, integral too large to integrate), 3.98 (s, 4H), 4.18 (bd m, 4H), 5.91 (bd m, 2H;), 7.48 (bd m, 2H), 7.76 (d, 4H), 7.88 (d, 4H), 9.38 (bd m, 2H): iodoacetyl determination (European Journal of Biochemistry 1984, 140, 63–71): Calculated, 0.46 mmol/g; Found, 0.37 mmol/g.
Conjugation of Peptides to Valency Platform Molecule #3 or #5:
All buffers were sparged with helium. The polyethylene glycol (PEG) derivative, #3 or #5 was dissolved in a minimal volume (approximately 20 mg/mL) of 0.05 M NaHCO3 (pH 8.25). Approximately 3 equivalents of peptide were used per iodacetyl group on the PEG derivative. For para-aminobenzoic acid (PABA)-PEG, containing 2 iodacetyl groups (MW=approximately 4100 g/mole), 6 equivalents of peptide were used for each equivalent of PABA-PEG. For diaminobenzoic acid (DABA)-PEG, containing 4 iodoacetyl groups (MW=approximately 4300 g/mole), 12 equivalents of peptide were used for each equivalent of DABA-PEG. The PEG solution was added to the reduced peptide solution and allowed to react for at least one hour in the dark. The peptide conjugate was purified by preparative HPLC. Before pooling and lyophilization, fractions were checked by electrophoresis using a 15% tricine gel. A description of the compositions of the five peptide conjugates is given in Table 3 and the structures are shown in
Murine Lymph Node Proliferation Assays.
Food and water was provided ad libitum. Balb/c mice were immunized in each hind footpad with 50 μg of melittin molecule in CFA. Popliteal lymph nodes were harvested aseptically seven days later. Lymph nodes were gently dissociated by teasing the cells through a 50 mesh sieve screen. The single cell suspension was washed in RPMI-1640 (Irvine Scientific, Irvine Calif.) containing glutamine, penicillin and streptomycin. 5×105 cells in RPMI medium supplemented with 10% fetal bovine serum (FCS) in quadruplicate wells of round bottom 96-well Corning microtitration plates were cultured with melittin or a melittin peptide at 10, 1.0 or 0.1 μ/mL. Cells in the positive control wells were cultured with murine interleukin 2 (IL-2) at 100 or 50 U/mL, PHA (phytohemagglutinin) at 1 μg/mL. The negative control wells contained lymph node cells in RPM-1640 and 10% FCS. The cells were cultured for 4 days in a 37° C. incubator with 5% CO2. Each well was pulsed with 1 μCi of [3H]thymidine (ICN, Biochemicals, Costa Mesa, Calif.) for an additional 18 hours. Cells were harvested onto a glass fiber filter mat using a semiautomatic cell harvester (Scatron, Sterling, Va.). Incorporation of [3H]thymidine was determined by liquid scintillation. The results were expressed as average counts per minute.
In Vivo Protocols
Balb/c mice were primed intraperitoneally (i.p.) with 4 μg of melittin in CFA. One month later the potential tolerogen or formulation buffer was administered i.p. Three days later all mice received an i.p. injection of 4 μg of melittin in Incomplete Freund's Adjuvant (ICF) (Sigma Chemical Co., St. Louis, Mo.). 100 to 200 μL of blood was collected from the retro-orbital venous plexus 10 days later. Serum samples were assayed for anti-peptide, or anti-melittin, IgG antibodies.
Assay for IgG Anti-Melittin or Anti-Melittin Antibodies
An individual mouse's serum sample was assessed serially for the presence of anti-melittin antibodies by ELISA. Falcon Probind 96-well microtitration plates were precoated with 10 μg/mL melittin or melittin peptide in phosphate buffered saline (PBS), pH 7.2, overnight at 4°. The plates were washed twice with a wash solution containing PBS, 0.02% Tween-20, and 1% gelatin (Norland Products Inc., New Brunswick, N.J.). Plates were blocked with 200 μL PBS containing 5% gelatin for 1 hour at 37°. Serum samples were prepared in a diluent of PBS containing 5% gelatin. Samples were tested at dilutions of 1:100 to 1:1000. After 1 hour of incubation at 37° C., the plates were washed four times. ExtraAvidin peroxidase (Sigma Chemical Co., St. Louis, Mo.) was diluted 1:1000 in PBS containing 5% gelatin. The plates were incubated 30 minutes at 37° C. and then washed five times. Wells were developed with OPD, (ortho phenylene diamine dihydrochloride, Sigma Chemical Co., St. Louis, Mo.) according to the manufacturer's directions, in the dark for 15–30 minutes, and the reaction was stopped with 3 M HCl. The optical density (OD) was determined at 450 nm on a microplate reader (Bio-tek Instruments Winooski, Vt.).
Antibody Forming Cell Assay
Cellulose microtitration plates (Millipore Co., Bedford, Mass.) were prepared as indicated above for the IgG antibody (ELISA) assay. However, at the point in the assay where the serum samples were added to the wells, splenic cells (5×105/well) were added instead of serum, and incubated overnight. The remainder of the ELISA assay was performed as indicated above.
T Cell Epitopes
T Cells from mice primed with melittin showed T cell proliferation in response to the whole melittin molecule and to C-terminal melittin peptides 3, 4, and 5 (
Female C57BL/6 mice, ages 5 to 8 weeks were purchased from The Jackson Laboratory, Bar Harbor, Me. Animals were maintained and treated accordingly to National Institutes of Health guidelines.
Immunization Protocol
Mice were primed by an i.p. injection containing 5 μg of melittin precipitated on alum and 2×109 B. pertussis as an adjuvant. The mice were boosted with 5 μg of melittin, i.p., in PBS.
pfc Assay
Sheep Red Blood Cells (SRBC) (Colorado Serum Co., Denver, Colo.) were conjugated with melittin-peptide 2 using carbodiimide. Fresh SRBC (less than 2 weeks old) were washed four times with cold saline and one time with mannitol (0.35 M mannitol, 0.01 M NaCl). The SRBC were suspended in mannitol to a concentration of 10% (v/v). 100 μL of mannitol containing 30 μg of melittin peptide #3 were added to 1 mL aliquots of 10% SRBC which were then incubated on ice for 10 minutes. 100 μL of a 100 mg/mL solution of 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide HCl (EDCI) was then added and incubated on ice for 30 minutes. The SRBC were washed twice with Balanced Salt Solution (BSS) (Irvine Scientific Co, Irvine, Calif.) and resuspended, to 10% (v/v). Lyophilized guinea pig complement (GIBCO, New York, N.Y.) was reconstituted with BSS and then diluted 1:3 with BSS. One mL of the diluted guinea pig complement was added to 3 mL of conjugated SRBC. Rabbit anti-mouse IgG was added to give a final dilution of 1:100 of the rabbit antiserum. This concentration was predetermined to inhibit all IgM pfc while enhancing the maximum number of IgG pfc. An equal volume of this complement/anti-mouse IgG/SRBC suspension was mixed with a cell suspension of mouse spleen cells taken from a single mouse. 50 μL of each mixture was transferred to the chambers of a Cunningham slide (three chambers per slide). The edges were then sealed with paraffin and incubated at 37° C. for one hour. The number of plaques per chamber was counted with the aid of a dissecting microscope. Each spleen suspension was also assayed using non-conjugated SRBC as a control. The number of viable cells, in each spleen cell suspension, was determined. The number of pfc per 106 spleen cells was determined for each chamber and the mean of the triplicates calculated. The number of pfc for non-conjugated SRBC was subtracted from the number of pfc for conjugated-SRBC to determine the number of peptide-specific pfc.
Determining The Optimal Time to Measure pfc
Mice were primed with melittin. Groups (3 mice per group) of primed mice were boosted with melittin on days 2, 4, 6, and 8. On day 10 the mice were sacrificed and their spleens harvested. Cell suspensions were prepared and assayed for the number of peptide specific pfc determined. The optimal number of pfc was obtained 6 days after boosting with melittin.
The Orientation of The Peptide on The PEG Conjugate Does Not Affect The Conjugate's Ability to Induce Tolerance
Two different tolerogens were constructed to determine if the orientation of the peptide on the PEG conjugate affects its ability to induce tolerance. Peptide #7 (equivalent to peptide #2 plus C-terminal penultimate insertion of Lys-Cys) was covalently bound to valency platform molecule 3 through its C-terminal end to make melittin Conjugate 3. Groups, (3/group) of mice primed with melittin were treated, i.p., with conjugates or with saline. Five days later all of the mice, including the non-treated control group, were boosted with 5 μp of melittin. Six days later the mice were sacrificed, their spleens were harvested and the number of peptide specific pfc determined. As illustrated in Table 4, both orientations were effective in reducing the number of peptide-specific pfc/106 spleen cells in mice primed and boosted with the parent protein melittin.
Three different conjugates, each with a different number, of peptides per PEG conjugate, were constructed to determine if the ratio of peptides to PEG molecule was important. Conjugate 1 had only two peptides per PEG conjugate. Another had four peptides per PEG conjugate (Conjugate 2). The third had four, branched peptides (8 B cell epitopes) per PEG conjugate (Conjugate 5). Groups (3/group) of mice primed with melittin were treated, i.p., with the different conjugates or with saline. Five days later all of the mice, including the non-treated control group, were boosted with 5 μg of melittin. Six days later, the mice were sacrificed, their spleens were harvested and the number of peptide-specific pfc determined. As shown in Table 5, Conjugate 1, containing two peptides per PEG molecule, was ineffective in reducing the number of peptide-specific pfc/106 spleen cells in mice primed and boosted with the parent protein melittin. The results show that both melittin conjugates 2 and 5 were effective as tolerogens; however, conjugate 5 which contained 8 epitopes (4 branched peptides) was effective at a lower dose than conjugate 2, which contained four unbranched peptides per valency platform molecule.
Modifications of the above-described modes for carrying out the invention that are obvious to those of ordinary skill in the fields of immunology, chemistry, medicine and related arts are intended to be within the scope of the following claims.
Patent Citations
Non-Patent Citations
Referenced by
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text:
It gets my goat.
The way people write the millions of gold as 1000k.
It's as ridiculous to my eyes as 1337speak.
Mainly because I read 1000k as "One thousand-thousand"
It's technically right. But it's an arse-about way of saying One Million.
How hard is it to type 1m? It's even less typing for the typing impaired!
Does it make it seem like more?
Would you rather have "A thousand thousand gold" or "ONE MILLION GOLD"
Anyway, rants do belong in journals, so this is where you find it. XD
Have a nice whatever it is you're having right now!!!
Community Member

subset: commoncrawl


text:
Posts with tag "video"
Billy Ray Cyrus on Miley's Lap Dance: 'It's What People Her Age Do'Posted May 13th 2010 at 09:10 PM by PopEater Staff
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Yesterday, TMZ unveiled video of Miley Cyrus dirty dancing at last summer's wrap party for her most recent flick, 'The Last Song.' The video shows the pop star giving the movie's producer, 44-year-old Adam Shankman, a pretty intense lap dance. And today, TMZ caught up with her dad to get his thoughts on his daughter's graphic dance moves. The video shows openly gay Shankman grinding up against ...
Tila Tequila to Try Adopting Casey Johnson's BabyPosted Jan 13th 2010 at 04:32 PM by Chris Jancelewicz
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- TV News -
- Videos
Just when you thought Tila Tequila had finally squeezed the last drop of fame out of her 15 minutes in the spotlight, the reality starlet has now told TV's 'Extra' that she's going to attempt to adopt the late Johnson & Johnson heiress Casey Johnson's baby. In the interview with 'Extra', Tila claims that Johnson's last wish "was to have [Johnson's daughter] Ava, have me have Ava."...
Paula Deen Takes a Ham to the FacePosted Nov 24th 2009 at 08:34 AM by PopEater Staff
Note to Paula Deen: Always beware of flying pork. The Food Network celebrity chef was trying to do some good by volunteering for the pre-Thanksgiving Hosea Feed the Hungry food drive in Atlanta, and before she new it, she was hit square in the face (nose and mouth, mostly) by an airborne ham that was being tossed for loading. Deen, clearly shaken by the incident, holds her face and is escorted o...
Allison Iraheta 'The Shoot' Video ExclusivePosted Oct 28th 2009 at 08:18 AM by PopEater Staff
- Filed under:
- American Idol -
- Music News -
- Videos -
- Exclusives
'American Idol' alum Allison Iraheta is ready to take the world by storm with her rock-laced tunes now that she's on her own. PopEater proudly premiered her single 'Friday I'll Be Over U' earlier this month, and now we've got exclusive behind-the-scenes footage from her album photo shoot. In it, Iraheta is seen getting done up in rocker chic, looking, as she proudly says, "awesome." Check It Out A...
All-American Rejects' 'I Wanna' VideoPosted Oct 13th 2009 at 06:24 AM by PopEater Staff
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- Music News -
- Videos -
- Exclusives
The All-American Rejects are anything but the latter portion of their name, having accrued a massive catalog of hits and legions of fans in their short time since breaking through, and now PopEater is exclusively premiering the video for the band's new single, 'I Wanna.' Whether it's their initial hit 'Swing, Swing' or their recent smash 'Move Along,' you know what you're going to get when AAR sta...
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text:
I will not be in church tomorrow
If you visit me in my modest apartment tomorrow morning, I will not be in church.
I will be enjoying myself with a bottle of red wine – baron de La Laguna – courtesy of my workaholic egbon, friend and colleague, Mr. Tunji Adegboyega, who has just been meritoriously elevated as Vice Chairman of this newspaper's Editorial Board. It is most unlikely that I will be reading the Bible either, that invaluable treasury of spiritual and intellectual illumination. No, I will most likely be engrossed in Wole Soyinka's new masterpiece "Thus spake Orunmila" based on the second book of Samuel in the Bible. Jesus! If the Nobel can be won twice, this man deserves it. This work fascinates. It titillates. It scintillates. It mesmerizes. It entices. It seduces you to transverse uncharted territories of the mind and human imagination.
But unlike Woke Soyinka, I am not a devotee of Ogun., the fabled Yoruba god of iron. I do not share the great intellectuals' disdain for the authority of divinity. I believe that I am only because God is. I believe not just in the historicity of Jesus Christ but in the enduring reality of his eternal presence. But then, why will I not be in church tomorrow? The answer is simple. I cannot understand the intense religiosity of a nation and the vulgar prosperity of its church amidst the abject material and spiritual poverty of its people. It is a study in contracts. Many people deposit their brains at home before proceeding to church on Sundays. Mine is a rather problematic organ. It insists on following me about.
For instance, I used to worship in a church at Oregun. I love the handsome pastor. His jerry curls never cease to dazzle me. Much more importantly, his insightful adumbrations of the word of God always excite me. But I suddenly discovered that he is hardly ever around. Half of the time during service is devoted to watching videos of his 'miraculous feats' abroad. I am told that he resides more in South Africa these days. The Holy Spirit may have informed him that the South African currency is more prosperous than the Nigerian Naira. Shout Halleluyah somebody. But if I wanted to watch video, couldn't I as well have done it at home?
There is another church where I loved to worship at Maryland. I admire the pastor's humble disposition despite his sartorial elegance. His speech is eloquent. His dissection of the word is impeccable. Last year, during the Christmas Carol, he assembled a group of comedians led by the one and only Lepacious Bose to entertain our minds even if our spirits remained famished. But then, I am reluctant to return to that wonderful church. One Sunday his wife preached. She said she was led by the spirit to ask those who were willing to contribute at least one million Naira to the new church building, to come forward. Later, the spirit led her to reduce the amount to: five hundred thousand Naira only. This stupid brain of mine refused to stay at home. Why could such donations not be made anonymously my brain questioned? Was the goal to honour God or man?
On my last Sunday in the church, the man of God preached brilliantly. At the end of his unimpeachable sermon, he announced that the Senior Pastor would soon be clocking 60. "This is a man who has done so much for us" he said "and how can we say thank you"? My adorable pastor announced that it had been decided to buy a car worth N16.5 million Naira for the senior pastor complete with an Eagle at 60 symbol. Donors with no less than one million Naira each were asked to see the pastor after service. In fact, somebody had already pledged three million Naira the pastor said. Now, I happen to know that this particular senior pastor has a convoy of Hummer Jeeps. Why then waste 16.5 million Naira on buying him a vehicle in a country where hundreds of millions live on less than $1 dollar a day? Is this the spirit of Christ? I wonder.
I used to travel out of town to a neighbouring state to worship in a church presided over by a man I love and admire so much. When I heard the rumour that he actually slapped a girl in the course of deliverance, I dismissed it out of hand. I immediately called a friend of mine who is a lecturer at the church's university to clarify the details. He was evasive and hedgy. Last Thursday a colleague played the entire video to me. It was shameful. It was despicable. It was barbaric. As far as I know, an individual has the right under our laws, to be a witch. Although the cleric claims that the object of his physical assault has since come to apologise to him, no visual evidence has been provided to authenticate this assertion.
I believe strongly that the church has a mission to save a human civilization that is clearly adrift. It is, however, unfortunate that the church itself, especially its Pentecostal variant, that claims moral and spiritual superiority is lost in the wilderness. Most of our Pentecostal leaders today lust after such titles as Bishop, Archbishop, Prophet, Apostle or Doctor. Amazingly, Jesus was simply Jesus! Dear Reader, I will not be in church tomorrow. But if you need some exquisite red wine, please consult Mr Adegboyega.
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Akala and the looting of patrimony
A few days after losing his reelection bid, Governor Christopher Alao Akala of Oyo State, with his characteristic sardonic humour, enacted a law ostensibly to ... Full story


text:
Get Ready for Fall with Philosophy Shower Gels
Time for Fall kids which means the Philosophy Fall Favorites Shower Gel Trio should be gracing your shower shelves! Call me crazy but I rotate my shower gels each season so it is indeed time for me to take out my favorite Philosophy Fall Shower Gels!
Take a look at this trio available exclusively at QVC.com.


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You can now invite anyone to meet at your favorite Starbucks.
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26 year old woman | Champaign, IL, USA | Seeking men 25-35 within 50 miles
I love to laugh out loud, from the belly, deep laugh. A laugh a day keeps the doctor away... Or is that an apple? Things that make me smile are relaxing sunny days, people watching, an ice cold coke, a fruity wine concoction, my cat & dog, and popcorn while watching the big screen. I'm easy to please and excited about the smallest of life's pleasures. I am very shy at first but eventually turn into a stand up comic... modest, I know. I am looking for someone who is driven and responsible when need be, but also fun and spontaneous. Not too much to ask, right? Be a lover. Be a friend. Make a girl laugh and you will win her over!
Camping , Coffee and conversation , Cooking , Dining out , Hobbies and crafts , Movies/Videos , Music and concerts , Exploring new areas , Performing arts , Shopping/Antiques , Travel/Sightseeing , Volunteering , Wine tasting
Aerobics, Baseball, Dancing, Swimming, Walking / Hiking, Yoga
I exercise 1-2 times per week.
Don't display my sign
I have Cats, Dogs
Who doesn't love pets?
Middle of the Road
Baseball games, the beach (I'm from the east coast), I love traveling - I studied abroad in Ireland and was lucky enough to visit 10 countries in Europe while there. I would love to see more of our beautiful country :)
West Chester University of Pennsylvania, West Chester, PA
I love wine, country music, and reading. Whether you want to stay in or go out, I'm game. I like to learn about new places and people.
I love to go to baseball games, the movies, out to eat, and to the park on sunny days. I like doing just about anything. I'm really thrifty and like to do do-it-yourself crafts. I am not a huge partier, but I do like to go out occasionally.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'2" (157cms)
|5'2" (157cms) to 6'2" (187cms)
|Body type:
|About average, Athletic and toned, Stocky
|Blue, Green, Hazel
|Light brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Occasionally, Yes, but trying to quit
|Social Drinker
|Social Drinker, Moderately
|Nonprofit / Volunteer / Activist
|Labor / Construction, Legal, Education / Teacher / Professor, Nonprofit / Volunteer / Activist, Law enforcement / Security / Military
I work for a nonprofit agency that works with domestic violence &sexual assault victims. I have a dual bachelors degree in elementary education &early childhood education and also work at elementary school as a 1 on 1 aide for a student with autism.
|I'll tell you later
|$25,001 to $35,000, $35,001 to $50,000, $50,001 to $75,000, $75,001 to $100,000, $100,001 to $150,000, $150,001+
|Never Married
|Never Married, Currently Separated, Divorced
|Have kids:
|Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|Definitely, Someday
|White / Caucasian
|White / Caucasian
|Spiritual but not religious
|Christian / LDS, Christian / Protestant, Spiritual but not religious, Christian / Other
|Bachelors degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


text:
LTHW Light Hiking Socks Foot Protection with THOR•LON® and THOR•WICK® COOL fiber
The Design of Thorlos Padded Hiking Socks
What Independent Clinical Studies Say About Thorlos
Your Feet will Feel Better
or Your Money Back®
Designed for feet that hurt. Enjoying day hikes in warmer weather, on flat to varied terrain resulting in low to moderate stress on the feet. Specifically engineered to provide protection and comfort for women prone to blisters, foot pain, or discomfort.
- Constructed with THORLON® blended with THOR•WICK® COOL fibers for superior softness, resilience, durability and extra moisture-wicking for drier, cooler, better feeling feet.
- Cushioned instep and arch for a better fit, more support and less pressure on the feet for a more comfortable day hike.
- Engineered for women's feet with extra stretch yarn in the ankle, a smaller heel pocket and a comfort toe box for a better fit.
- Low profile toe seam won't rub or irritate the feet.
- Unique padding specifically engineered for light hiking, clinically shown to reduce blisters, pain, pressures and moisture.
- Ventilation panel for extra moisture control and breathability.
Fiber Contents | 19% Exclusive THOR•LON® Acrylic | 14% Stretch nylon | 6% Spandex | 61% THOR•WICK® COOL
Reviews for Thorlos LTHW
Customer Reviews
Very comfortable plus they stay UP!
05/25/15 | By Cynthia M.
I like that my feet stay warm in them, without getting too hot. The sock is more fitted than a typical sock, so ordering the correct size is important. I've worn them with hiking/work boots as well as with a pair of slip on shoes that were a little big. The socks made all the difference in comfort when wearing the two very different shoe types. My calves aren't skinny, and socks often slide down easily from being stretched. These have a nice grip, but never feel snug on my calf. I will definitely order more socks when I've saved up for them!
comfortable hiking sock
04/28/15 | By Karen l R.
good cushion, embraces the foot nicely. I would prefer not so tight around the upper part of sock.
Best socks for hiking!
04/25/15 | By Kathleen K.
Confident that I will not get blisters and that my feet will not get wet with sweat.
Great Hikers
04/25/15 | By Madelyn O.
I got to try these for free and I have since bought a few more pairs at REI! Great with my hiking boots and they don't feel too thick and constricting.
04/24/15 | By Jennifer C.
I am so glad I tried these socks! They are so comforting to my ever moving feet!
Best motorcycle socks ever
04/23/15 | By Tammy P.
I love wearing these with my Harley boots! They give my foot an all-around padded feeling that keeps my boots from rubbing my feet. It's the only socks I wear when riding my Harley!
Great for hiking
04/22/15 | By Sarah C.
These socks are very comfortable, my feet felt cushioned during hikes of 6-8 hours per day. My feet also felt cool and dry.
Great for Hiking
04/20/15 | By Sandy K.
I do wear a pair of thin socks with the Thurlos, but the combination is very comfortable for hiking and sweatty feet.
04/20/15 | By Pamela W.
Comfy socks that absorb a lot
Just tried them once - very comfortable.
04/18/15 | By Joannah M.
I feel like I have a comforting wrap around my foot. I almost never wear socks, but with athletic shoes, I need them. Going to walk the Camino de Santiago in September and October for the second time, and will take a pair of these with me, though I have other favorites from last time. We'll really give these the test! Thanks for the free offer. I heard about your socks from the American Pilgrims On The Camino Facebook page. Thanks again. Joannah Merriman
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1:56 pm
Thu May 13, 2004
50th Anniversary of Hank Williams' Death
By Alabama Public Radio
Tuscaloosa, AL – January 1st, 2003 was the 50th anniversary of the day legendary country music singer Hank Williams Senior died. Williams is still considered by many to be one of the greatest country music singers ever. Lance Kinney, host of Alabama Public Radio's Bama Bluegrass, reports on the life and music of Hank Williams Senior.
OUTRO: The preceding report, which originally aired January 1st, 2003, was written by Kathie Farnell and produced by Alabama Public Radio's news department. The report was supported in part by funding from the Alabama State Council on the Arts.


text:
Open Face Pita Sandwiches
23 May 2010
|Sun dried tomatoes in oil
|4 Ounce, drained and (1/2 Of 8 Ounce Jar)
|2 Tablespoon, reserved
|Skinless boneless chicken breast halves
|4 Small, cut into about 1/2 inch pieces
|Italian seasoning
|1⁄4 Teaspoon
|1 Small, thinly sliced
|Grated parmesan cheese
|1⁄4 Cup (4 tbs)
|Whole wheat pita breads
|2 (6 Inches In Diameter)
|Shredded mozzarella cheese
|2 Ounce (1/2 Cup)
Heat oven to 375°.
Heat reserved oil in 10-inch skillet over medium-high heat until hot.
Saute chicken, Italian seasoning and onion in oil about 4 minutes, stirring frequently, until chicken turns white.
Cut tomatoes into 1/4-inch strips.
Stir tomatoes and Parmesan cheese into chicken mixture.
Split each pita bread in half around edge with knife to make 4 rounds.
Divide chicken mixture evenly among rounds.
Sprinkle with mozzarella cheese.
Bake about 5 minutes or until cheese is melted.
Rate It
0 Comments
Open Face Pita Sandwiches Recipe


text:
Level 27. Again.
Duff_ManX Blog
Bionic Commando....again
by Duff_ManX on Comments
Well it seems I'm Bionic Commando for the second time around... so here goes:
Level 28: Bionic Commando
"Bionic Commando - ****c NES game in which you played as a bionic commando who could not jump, but had a nifty extending bionic arm that let you swing from stuff. Truly kick-ass."
The original Japanese version of Bionic Commando ("Top Secret: Hitler's Revival" - Japanese title) placed the main character, Radd/Ladd Spencer, in a battle against futuristic Nazis. The final boss was Adolf Hitler who had been resurrected by evil scientists. The story featured in the English version of the game begins with a search for Super Joe, who was the main character of Commando. In truth, Commando and Bionic Commando are actually unrelated games.
Unlike the arcade version, the NES version is an action/adventure game, in which the player has to explore each stage and obtain the necessary equipment to progress through the game.
Map: Instead of moving in a straight path through the levels, the player can, between levels, move around on a map-like screen. The player can move his helicopter one space at a time between the areas, represented by numbers.
Bionic arm: The main character is a commando with a bionic arm which can grab onto surfaces, allowing him to pull himself forward, or swing from the ceiling. The game is notable for being one of few platform games in which the player cannot jump. To cross gaps, the hero must swing like Tarzan on a vine.
^ Bionic arm in use ^
Health: Enemies can sometimes be defeated for "bullets", which, if the player gets enough of them, will add extra units of health. This is important because the player only starts with one life point.
Well I'm back. It's time to start off fresh.
by Duff_ManX on Comments
I have been banned for nearly 6 months but now I come back to you, a changed person.
I hope to see you all around.
The Moment We've All Been Waiting For...
by Duff_ManX on Comments
The Simpsons Movie Has Arrived!
I am the Karate Champ & PC Stuff
by Duff_ManX on Comments
Level 31 - Ippon!
A reference to Karate Champ, possibly the first arcade martial-arts game.
Karate Champ (original title: "The Way of Karate") is a 1984 arcade game developed by Technos Japan Corporation for Data East. It is one of the first fighting games and has been believed to be the first to use today's common side-perspective.
In this game, the player assumes the role of a Karate competitor and fights against another player or the computer. Using two joysticks, players can execute a number of moves.
Gameplay consists of a two dimensional fight between Karate characters wearing white and red gi, followed by various bonus rounds for the successful player. This pattern repeats itself in the next, more challenging round set against a new background. Unlike most later fighter-type games, there are no health bar or hit points. A hit successfully landed ends the round and earns the player or his opponent either one point or half point. The first to two points is the winner.
In other news...
I am getting my PC completely reformatted tomorrow and it will take a few days for everything to get set up again, so I most likely won't be on GS during that time. I hope you all can continue with your Unions and blogs as usual in my absence, and I'll try and get back on here as soon as I can. Peace!
- Duff_ManX
Moe's Tavern Official Union Video
by Duff_ManX on Comments
Here is the Official Union Video I have created for the Moe's Tavern Union. I hope you like it!
Please join if this interests you!
Why Lost is a great show...
by Duff_ManX on Comments
Here is all of the different perspectives shown of Oceanic Flight 815 crashing, from the TV series 'Lost'.
Viewpoints are from inside the plane (Pilot), along with Desmond not pressing the button (season 2 finale) and The Others watching from the ground (season 3 premiere).
Well worth a look for anyone remotely interested in why Lost is such a great show! Epic stuff. :)
Long time, no blog!
by Duff_ManX on Comments
Two topics today.
The first is a humorous piece of writing I found in a gaming magazine titled:
The Journos Who Ate San Francisco
"Take a single bite of a Jack In The Box burger, and not only will you instantly become obese, but in a violent hallucination, your life will flash before your eyes and you will finally known that there really is a Satan, and his name is Bacon Ultimate Cheeseburger."
And here is a funny scene from a recentish episode of The Simpsons:
Bye for now! :D
I am teh Wiki Sick
by Duff_ManX on Comments
Hm, this title has two meanings.
One, I am sick and tired of Wikipedia. All the nagging editors who whine on and on if you so much as make an edit or upload a file that they don't like. Some people seriously need to get a life.
Two, I have just leveled up to Level 30: Wicked Sick!
According to GameSpot, this is "The award earned in UT2k4 for 30 consecutive frags without a death."
Not much else more to say other than "Yay"! :D
Don't forget to check out my last epic
VV blog post directly below this one. It's nice! VV
Pokemon: Advanced GRAWfighter
by Duff_ManX on Comments
Ladies and Gentlemen, it's that time again.
OoMandaloreoO and I have been creating various concept designs in the .exe program known as Photoshop CS2. We are interested in your opinions for our up and coming feature film simply titled:
"Pokemon: Advanced GRAWfighter"
Please leave feedback about this soon to be blockbuster. :D


text:
Casey and his cart return to US Open
SAN FRANCISCO Casey Martin has never allowed himself to look too far into the future.
Even looking back, it is no less amazing to see him and his cart back at The Olympic Club, riding between shots during a practice round Monday at the U.S. Open, then walking painfully back to the cart with a limp that has become as much a signature for him as a fist pump for Tiger Woods.
Martin could not have predicted 14 years ago when he left the U.S. Open after his historic ride that he would still be competing against the best in the world. He gave up tournament golf six years ago and took over as golf coach for the Oregon Ducks.
He could not have predicted he would still even have a right leg.
"I'm 40 now, and so this is at that point where I didn't know if I would ever really be able to keep my leg," said Martin, who suffers from a rare circulatory disorder that led him to sue the PGA Tour for a right to use a cart. "So it's not great. When I wake up, I feel it. When I get out of the golf cart, I feel it. When I travel with the team and travel down here, I definitely feel it. That's always going to be the case. And so I'm not complaining. It's hanging in there.
"But I'm not going to be running a marathon, either."
Running a marathon seemed more plausible than Martin playing another U.S. Open - at Olympic Club, no less.
The only competition Martin has had over the past six years was an occasional game with his players, or a charity event that often featured a scramble format on short golf courses designed for amateurs. But with Olympic hosting another U.S. Open, he figured it was worth a shot.
King Martin recalls his son telling him he thought he would enter the U.S. Open.
"As a USGA member, I got a USGA hat in the mail," the father said. "I put it up in my office at home, let it sit there, clinging to that dream. It has a little more meaning to me right now. This is a godsend, I can tell you that."
It's a script even Hollywood would have a hard time believing.
His coaching schedule allowed Martin to go through local qualifying in Washington, and in his first serious competition since he became a golf coach, Martin made it through. The sectional qualifying last week was two days after Oregon reached the NCAA semifinals at Riviera. On little sleep, Martin was on his way to claiming one of two spots for the U.S. Open when he couldn't find his tee shot on the fifth hole of the second round.
His caddie found it at the last minute - it was hidden by a clump of mud, and Martin believes a cart was parked over the ball at one point - so instead of going back to the tee and probably taking double bogey, Martin hacked it short of the green and chipped in from 30 yards for birdie.


text:
The Long Way Home. Huntsville to Cleveland on a 250.
View Single Post
Beastly Adventurer
Joined: May 2008
Location: Huntsville, AL
Oddometer: 6,725
After Burke's garden I headed to the town of Tazewell. I just rode through taking one Pic as I rode.
Then I turned on to 16 South. Yes, I know that the way to Cleveland was north but one of the things I wanted to do on this trip was ride 16 between Tazewell and Marion. It's a great road that twists and turns its way up and over three mountain ridges. Since I was headed the wrong way on 16, I would have to ride that section twice
. Oh well, if someone had to do it, it might as well be me.
Just outside of Tazewell I came across this:
It's a chicken farm....or maybe ranch is a better term. Each chicken had its own little shelter and was on a leash. Definately different from the typical chicken farms we have in North Georgia.
Then it was some fun riding through a bunch of twisties. IMO, this section of 16 is much more challenging than the much more famous (infamous?) Deal's Gap.
I did stop for a break. I turned down a dirt road.
went a short distance and parked.
Good thing I was not in a passenger car
Of course what fun would driving this road be in a cage anyway
If I was on a dual sport I probably would have enjoyed seeing where this road went. There were a number of other dirt roads along 16 but this was the only one with a warning sign.
After crossing all three mountain ridges, I came to Hungry Mother State Park. The park may have a strange name but it sure looked like a nice park.
I;m not really much of a beach person. Beaches in Florida leave me bored after a couple of hours but there is something about rivers and lakes in the mountains that I find really beautiful. It was still too early to stop, especially since I already lost so much time getting a tire this morning. Despite this, I decided to stay here for the night. I had only ridden a little over 100 miles and was only about five miles north of where I spent last night but since I had no real plan, no reservations, and plenty of time, I decided I could afford to do this.
So I paid for a campsite. It was a little pricey;$21 plus a five dollar 'processing fee" whatever that was. It did include entry into the park and also the beach.
I went and found a campsite and set up my tent.
Then I headed off to the beach. Throwing caution to the winds, I rode that half mile to the beach in water shoes, T shirt, swim trunks, helmet and gloves.
I took a few pics then went swimming.
There was a platform with a couple of diving boards.
Did I mention it was very scenic here?
and more scenery:
The water was perfect, a little cold at first but nice and refreshing once I got used to it.
Then it was back to my campsite.
These campsites were unusual since all tents had to be on these wooden platforms. There was a nice bath house about 50 yards away and the area was on the side of a mountain and completely tree covered.
For dinner I rode into Marion and ate here:
Good food, good service and reasonable prices. I'll give the Tuscan Italian Grill a thumbs up.
Then I stopped at McDs for a milkshake and then back to the park. Total mileage for the day: 132. Total progress toward my destination: around 5 miles.
I ride, Therefore I Am.
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text:
Barkley snatches Sixways win
Bath claim the points in away victory at Worcester
Last Updated: 01/09/12 5:43pm
Olly Barkley kicked a last-minute penalty to have the last laugh in his kicking duel with Andy Goode as Bath edged out Worcester 24-23 at Sixways.
Goode looked to have won it for the hosts when he nailed a penalty - his sixth of the afternoon - with two minutes left, only for Barkley to trump him with his eighth successful three-pointer in the dying seconds.
The home side had raced out to a 12-0 with Goode initially solid with the boot as he slotted a 40-metre penalty in just the second minute.
Minutes later Bath offended in the ruck area and again the outside half was on target, this time from 35 metres.
The Warriors continued to dominate play and when Matt Banahan was penalised for not rolling away at a ruck, Goode converted his third penalty to give his side a 9-0 lead after only 10 minutes.
Worse was to follow for Banahan and Bath. The centre was yellow carded for a deliberate knock on and again Goode made no mistake with his penalty kick.
Banahan was soon joined in the sin-bin by rival captains, Stewart Hooper and Dean Schofield, who were yellow carded by young referee, Luke Pearce, after a substantial melee involving most of the forwards.
Barkley reduced the arrears with the first of his eight penalties and Banahan then returned with no damage done to his side's chances.
Another error from the Warriors saw Josh Drauniniu fail to collect an up-and-under and another penalty was conceded for offside which Barkley kicked from 45 metres.
A simple penalty from Goode after 32 minutes made it 15-6 to the Warriors and they should have extended that lead when they blew the best try scoring opportunity of the half.
Goode, David Lemi and Josh Matavesi combined brilliantly in a sweeping 70-metre move but Lemi's scoring pass to Goode was ruled forward.
Bath scored the first points of the second half when Barkley was successful with a 45-metre penalty and the lead was soon down to three points with another simple goal from the outside half.
The Warriors conceded another penalty at the ruck area to allow Barkley to level the score before he gave the visitors the lead for the first time in the match with a 35-metre penalty after 65 minutes.
Stung by the reverse Worcester immediately brought on three replacements and it soon paid dividends when Percival drove over from close range but Goode's conversion rebounded back of a post which proved costly as Barkley was successful with his longest penalty of the game to give Bath a one-point lead with six minutes remaining.
Back came the Warriors to secure two penalties in the Bath 22, Goode surprisingly missed the first but kept his nerve to kick the second, although Barkley made no mistake with his eighth penalty and with it an away victory.


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There's No Disgrace Like HomeThere's No Disgrace Like Home (1990) Season 1 Episode 7G04- The Simpsons Cartoon Episode Guide by Dave Koch
This page has been viewed 26 times this month, and 845 times total.
- Klasky Csupo, Gracie Films.
- Featuring: Bart Simpson, Homer Simpson, Marge Simpson, Lisa Simpson, Maggie Simpson, Barney Gumble, Tom Gammil, Receptionist, Mr. Burns, Waylon Smithers, Documentation Voice, Boxing Announcer, Eddie, Dr. Marvin Monroe, Pawnbroker, Mr. Gammil, Moe Szyslak, Lou, Daughter, Father in Monroe Ad, Mother in Monroe Ad, Son in Monroe Ad, Voice in Monroe Ad, Mothers, Fathers, Sons.
- Directed By Gregg Vanzo, Kent Butterworth.
- Produced By Richard Sakai, James L. Brooks, Matt Groening, Sam Simon, Al Jean, Mike Reiss, Larina Jean Adamson, Paul Germain, J. Michael Mendel, Craig Knizek; more Producers ...
- Animated By Ken Bruce, Craig Clark, Ed De Mattia, Brett Haaland, Mike Gerard, Eduardo Olivares.
- Written By Al Jean, Mike Reiss.
- Originally Released in 1990.
- Season: 1
- Color
There's No Disgrace Like Home Cartoon Pictures:
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