Fluorescence technique to demonstrate creatine phosphokinase isozymes.
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Animals↗
Biomedical subjects
G R Siber
Publications and source records attributed to G R Siber.
Determination of protein loading in biodegradable polymer microspheres containing tetanus toxoid.
Various methods to determine loading of vaccine in biodegradable polymer microspheres encapsulating tetanus toxoid were evaluated. The microspheres were composed of poly (D-lactic acid) (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA). Dissolution of microspheres in organic solvents such as methylene chloride, chloroform, or dimethyl sulfoxide and extraction of vaccine antigen or total protein with phosphate buffered saline gave variable results which depended upon the characteristics of the microspheres, such as type of polymer, excipients used in the microspheres and formulation conditions. Microspheres made from low molecular weight PLGA polymer and showing a large burst release exhibited up to 25% extraction of antigen whereas microspheres made from PLA microspheres with low burst release showed < 1% extraction. Extraction of total protein with 0.1 N NaOH and 5% sodium dodecyl sulfate showed results similar to those obtained with organic solvent extraction method. Partial digestion of microspheres with 6 N HCl at 60 degrees C for 20 h resulted in approximately 30% loss in TT protein by micro-bicinchoninic acid (BCA) assay. The major problem with this method was strong reactions in the micro-BCA assay of stabilizers, particularly sugars (glucose, sucrose) used in the microsphere formulations. Complete digestion of microspheres with 6 N HCl at 110 degrees C for 20 h or with 13.5 N NaOH at 121 degrees C for 1 h and quantitation of amino acids by a modified ninhydrin assay showed reproducible results on the protein loading in the microspheres. However, this method was affected by the presence of stabilizers, such as gelatin, which contain amino acids. Further, sucrose concentrations higher than 10% caused interference in the ninhydrin assay on samples hydrolyzed with 6 N HCl. In contrast, hydrolysis with 13.5 N NaOH did not show any interference by sucrose. Stabilizers used outside the microspheres for lyophilization purposes may be removed by washing the microspheres before loading determination or by dialysis but stabilizers used inside the microspheres would still cause interference. For reliable determination of total protein in the microspheres containing vaccines, we suggest complete digestion of microspheres with acid or base followed by amino acid analysis by colorimeteric assays such as ninhydrin method or using amino acid analyzers. The method needs to be optimized for each type of formulation to eliminate interference by the excipients. Alternatively, total protein nitrogen in the microspheres may be determined by the Kjel-dahl method if no amino acids or other nitrogen containing stabilizer is used inside the microspheres.
Antigens, Bacterial↗
Differences in the immunogenicity of native and formalinized cross reacting material (CRM197) of diphtheria toxin in mice and guinea pigs and their implications on the development and control of diphtheria vaccine based on CRMs.
Immunogenicity of native and formalinized cross reacting material (CRM197) of diphtheria toxin (DTx) was assessed in mice and guinea pigs. For the primary response, mice produced similar levels of diphtheria toxoid (DTxd) IgG antibodies to both native and formalinized preparations of CRM197 though the antibody levels were significantly lower than those elicited by conventional DTxd (P < 0.05). In contrast, guinea pigs showed significantly higher levels of DTxd IgG antibodies to the formalinized CRM197 preparation than the native preparation (P < 0.001) after single injection. These differences in the immunogenicity of mice and guinea pigs to native and formalinized CRM197 preparations have implications for the development and control of diphtheria or other vaccines involving the use of CRMs.
Animals↗
Antibody response of patients with Hodgkin's disease to protein and polysaccharide antigens.
We examined the antibody response of patients with Hodgkin's disease to a variety of vaccines to formulate guidelines for immunization. During and after treatment for Hodgkin's disease, both pre- and postimmunization levels of antibody to Streptococcus pneumoniae, Hemophilus influenzae type b, and tetanus toxoid antigens were significantly lower in patients than in controls. Impairments in the antibody response were most severe in intensively treated patients and improved as the interval between treatment and immunization increased. The primary, but not the secondary, antibody responses to the hemagglutinins of the influenza virus A/Victoria/75 and A/New Jersey/76 also were impaired in treated patients. Before treatment antibody responses to pneumococcal vaccine was normal regardless of the stage of disease unless treatment began within 10 days of immunization. Levels of antibody decreased during therapy in proportion to the intensity of treatment but remained higher than levels in comparably treated patients who were not immunized at diagnosis. We recommend that patients with Hodgkin's disease receive pneumococcal vaccine at diagnosis at least 10 days before initiation of treatment. Patients who are treated before immunization may be immunized several months after treatment, although the response of heavily treated individuals to vaccination may be marginal. More studies are needed to determine whether reimmunization of patients initially immunize at diagnosis is safe and effective.
Adolescent↗
Pharmacokinetics of intravenous trimethoprim-sulfamethoxazole in children and adults with normal and impaired renal function.
Thirty-seven children and adults aged 0.2-82 years were treated intravenously with 150 mg of trimethoprim (TMP) and 750 mg of sulfamethoxazole (SMZ)/m2 every 8 hr, usually for known or suspected pneumocystis pneumonia; when necessary dosage was adjusted to maintain peak TMP levels of 5-10 micrograms/ml. On day 2 of treatment, mean peak levels of TMP-SMZ were 7.02 and 148 micrograms/ml, respectively, and mean half-lives were 9.6 and 10.7 hr, respectively. All age groups achieved similar peak levels of TMP-SMZ, although dosages per weight were higher in children than in adults. Peak increments (peak levels minus levels before infusion) were higher and more reliable after iv than after oral dosage (P less than 0.001). The half-lives of TMP and SMZ increased with age (r = +0.73 and +0.39, respectively) and were correlated directly with the level of serum creatinine (r = +0.85 and +0.39, respectively). Serum concentrations of N4-acetyl-SMZ, the major hepatic metabolite of SMZ, increased in proportion to concentrations of creatinine in serum (r = +0.92; P less than 0.001). Adverse effects included fluid overload due to the large dilution volume and thrombocytopenia, which was associated with higher serum TMP levels and longer treatment as compared with nonthrombocytopenic patients. A loading dose of 250 mg of TMP and 1,250 mg of SMZ/m2 is recommended, followed by maintenance doses of 150 mg of TMP and 750 mg of SMZ/m2 every 8 hr for children aged 10 years or younger and every 12 hr for adults with normal renal function. In renal failure the dosage interval (hr) should be increased to 12 times the serum creatinine level (mg/dl) (maximum, 48 hr). Serum concentrations of TMP and perhaps of N4-acetyl-SMZ should be monitored in patients with severe renal failure.
Administration, Oral↗
Use of trimethoprim-sulfamethoxazole to prevent bacterial infections in children with acute lymphoblastic leukemia.
We assessed the efficacy of prophylactic antibiotics in children receiving intensive chemotherapy for acute lymphoblastic leukemia. The patients were randomized to receive either trimethoprim-sulfamethoxazole (TMP-SMX) or placebo in a double-blind trial. Thirty patients were evaluated in each group. Children receiving TMP-SMX had fewer episodes of bacteremia (0 vs. 5) and otitis media (3 vs. 18). The geometric mean of the neutrophil nadir was 172 in the TMP-SMX group and 287 in controls. However, no increased delay or dose reduction of chemotherapy was observed in the TMP-SMX treated patients. Five patients who received TMP-SMX developed Gram-negative rods resistant to TMP-SMX on surveillance stool cultures. We conclude that TMP-SMX prophylaxis decreased certain bacterial infections in children with acute lymphoblastic leukemia without causing clinically significant toxicity. The emergence of Gram-negative rods resistant to TMP-SMX in treated patients suggests that TMP-SMX prophylaxis should be restricted to patients who are at high risk for developing a bacterial infection or Pneumocystis carinii pneumonia.
Administration, Oral↗