In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration.

Parallel synthesis and biocatalytic amplification of a cross-conjugated cyclopentenone library.

A combination of parallel chemical synthesis and biocatalysis has been used to prepare and amplify a library of cross-conjugated cyclopentenones. A number of marine and terrestrial natural products with antibiotic activity are known to incorporate this pharmacophore. The library was screened for anticancer, antimycobacterial, antifungal, and antibacterial activity. The positive results from the screens provide an indication of the structural features that are associated with activity in the various assays and suggest promising avenues for further inquiry.

Cytokine production in cell culture by peripheral blood mononuclear cells from immunocompetent hosts.

The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.

Gamma delta T lymphocytosis associated with common variable immunodeficiency.

We present the case of a 28-year-old Caucasian female with common variable immunodeficiency (CVID) since age 5 who had a long history of hospitalizations for unexplained fevers and pulmonary infiltrates. The patient developed mild lymphocytosis 7 months prior to our evaluation. Flow cytometry of peripheral blood revealed an expansion of gamma delta T lymphocytes, mild CD4 T lymphocytopenia, and a reduced CD4/CD8 ratio (0.2). Two subpopulations of gamma delta T lymphocytes were found (CD3+/CD4-/CD8+, 47%; CD3+/CD4-/CD8-, 53%), the vast majority of which expressed V-delta 1. An infectious cause for the patient's gamma delta T lymphocytosis could not be found. The sputum was chronically colonized with Staphylococcus aureus, and the organism produced TSST-1 in vitro. A bronchoalveolar lavage (BAL) revealed marked lymphocytosis, but gamma delta T lymphocytes were not overrepresented in the BAL. Lymphocyte functional studies revealed poor proliferative responses to mitogens and staphylococcal superantigens and diminished cytokine production. V-delta 1 T lymphocytes from the patient's blood were not expanded in vitro in response to staphylococcal superantigens. TCR gene rearrangement studies confirmed the presence of J gamma and J beta 1 clonal rearrangements accounting for only a small subpopulation of the gamma delta T lymphocytes. These studies were repeated 5 months later and were unchanged. A bone marrow biopsy was negative for leukemia. Hence, the cause of the patient's gamma delta T lymphocytosis could not be determined despite evaluation for underlying malignancy, occult infection, or superantigen-driven stimulation. The patient ultimately died of progressive respiratory insufficiency. The state of current knowledge regarding gamma delta T lymphocytosis, decreased production of alpha beta T lymphocytes, and a low CD4/ CD8 ratio in association with CVID is discussed.

Comparative study of neutrophil activation by chemiluminescence and flow cytometry.

Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.

Chemiluminescent and flow cytometric analysis of gamma interferon preincubation on neonatal and adult rat polymorphonuclear leukocytes.

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.

Evaluation of the throat culture as a follow-up for an initially negative enzyme immunosorbent assay rapid streptococcal antigen detection test.

In a study of the efficacy of following up an initially negative enzyme immunosorbent assay (EIA) rapid streptococcal antigen detection test with a throat culture, 2 double swabs (4 total) were obtained from 264 pediatric patients with sore throats. Although a throat culture was more specific (97%) than the EIA (89%), the sensitivity (87%) and negative predictive value (97%) of a single EIA was the same as that of a single throat culture. A follow-up throat culture was more accurate than a follow-up EIA. We conclude that the office EIA tested results in more false positives but misses no more true positives than a single throat culture processed by a well-controlled microbiology laboratory. If a follow-up technique is used for an initially negative EIA rapid streptococcal antigen detection test, the throat culture is the superior test and would be equally applicable following an initially negative throat culture or EIA.

Protective mechanism of the immune response to a ribosomal vaccine from Pseudomonas aeruginosa. I. In vivo protection studies in compromised animal models.

Studies of the protective activity of a ribosomal vaccine from Pseudomonas aeruginosa in various immunocompromised animal models were performed. The results obtained demonstrated that the vaccine was highly effective in complement (C5)-deficient mice, C3-deficient (cobra venom factor-treated) mice, and leukopenic mice in providing protection against lethal infection with P. aeruginosa. Passive immunization with specific antiserum to the ribosomal vaccine was also effective in leukopenic mice. In mice that were both C5-deficient and leukopenic, the vaccine did not protect mice against lethal infection, but did prolong their survival, whereas in C3-deficient, leukopenic mice significantly enhanced protection was observed. These results were interpreted to suggest the involvement of multiple factors in the protective immune response to the vaccine, including the bactericidal and opsonic activities of specific antibody plus complement and the phagocytic activity of polymorphonuclear leukocytes with opsonized bacteria. Compensation for deficiencies in some of these factors can be obtained by enhancement of other factors through active or passive immunization.

Protective mechanism of the immune response to a ribosomal vaccine from Pseudomonas aeruginosa. II. In vitro bactericidal and opsonophagocytic studies with specific antiserum.

In vitro bactericidal and opsonophagocytic assays were performed with mouse antiserum to a ribosomal vaccine from Pseudomonas aeruginosa. The results obtained demonstrated that both specific antibody and complement are required for bactericidal and opsonic activities. C4-deficient complement also supported these activities. Polymorphonuclear leukocytes (PMNL) are not required, but when present, augment the observed killing activity. Opsonic and bactericidal activities were found to increase with increasing complement concentrations in an apparently logarithmic fashion until maximal activity was obtained. On the contrary, determination of bactericidal activity as a function of specific antiserum concentration revealed a "prozone" effect; i.e., maximal activity was obtained at an intermediate concentration of antiserum with significantly decreased activity at both higher and lower concentrations. This effect was not observed when immunoglobulin G (IgG) purified from specific antiserum by a method involving exposure to pH 9.0 and pH 3.0 was used instead of whole antiserum. In this case bactericidal activity increased as IgG concentration was increased, also in an apparently logarithmic manner.

Opsonic activity of antisera to ribosomal vaccine fractions with live and formalinized Pseudomonas aeruginosa.

The opsonic capacity of antisera to Pseudomonas aeruginosa ribosomal vaccine fractions was determined by a chemiluminescent technique. Antiserum to a vaccine fraction ("peak A") containing lipopolysaccharide (antiserum A), and antiserum to a vaccine fraction ("peak B"), which did not contain detectable amounts of lipopolysaccharide (antiserum B), were used to opsonify live or formalin-treated bacteria. Polymorphonuclear leukocytes were then stimulated by the opsonified bacteria in the presence of the chemiluminigenic probe, luminol, resulting in the observed chemiluminescence. The data obtained indicated that the antisera had comparable opsonic activity with live (untreated) bacteria. However, antiserum B had far less opsonic activity than did antiserum A when formalinized bacteria were used. Owing to the effects of formaldehyde on protein, these results were interpreted as evidence to suggest that the opsonic activities of the two antisera are dependent on different antigens on the bacterial cell surface. Antiserum A activity is probably dependent on lipopolysaccharide to a great extent, whereas antiserum B activity is most likely dependent primarily on a protein(s).

Active and passive immunization with Pseudomonas aeruginosa ribosomal vaccines and antisera in the burned rat model.

Pseudomonas aeruginosa ribosomal vaccines were tested for their ability to protect rats subjected to a 20% total body surface burn against the lethal effects of infection with homologous organisms. When administered prior to burning, the vaccines provided 100% protection. When administered postburning, the vaccine from one strain also provided 100% protection when the time interval between vaccination and infection was 3 days. When this time interval was reduced to 1 or 2 days, approximately 50% protection was obtained with the same vaccine. The vaccine from a second strain tested provided about 50% protection with a 3-day time interval. In addition, passive immunization using antiserum to a ribosomal vaccine was also demonstrated to be effective in protecting burned and infected rats, especially when multiple doses of antiserum were used. In this case, 80% protection was obtained (with no protection observed using multiple doses of normal serum). Finally, a comparison of ribosomal and lipopolysaccharide vaccines and antisera was also performed.

Kinetic analysis of microbe opsonification based on stimulated polymorphonuclear leukocyte oxygenation activity.

With Pseudomonas aeruginosa as the target microbes and polymorphonuclear leukocytes (PMNL) as effector phagocytes, the microbe-specific, immunoglobulin G (IgG)-dependent opsonic capacities of preimmune and immune sera were measured as the rate of stimulated PMNL dioxygenation of luminol yielding chemiluminescence (CL). When the reactants other than opsonin are present in concentrations that are not rate limiting, the information-effector relationship linking specific opsonin concentration to effector PMNL stimulation is described by the rate equation: L' = k'[IgG]i, where L' is the peak CL velocity (photons per minute), k' is the proportionality constant, [IgG] is the concentration of specific opsonin, and the exponent i is the order of the reaction with respect to opsonin. Since the specific opsonins were polyclonal IgG of unknown absolute serum concentration, the reciprocal rate expression, L' = k'D-i, was employed for data presentation; D is the serum dilution (final volume/initial serum volume), and the sign of i is changed to negative. The relationships of integral, first-derivative, and second-derivative expressions of the CL response to opsonin concentration are illustrated with experimentally obtained data. Based on peak CL velocity or peak CL acceleration measurements taken over different time intervals of testing, the estimated order with respect to opsonin is highest, and probably most accurate, using the shortest test interval allowing reasonably good precision of measurement. As an alternative temporal approach, microbe opsonification kinetics are analyzed based on nodal time (Tn) measurements. The Tn is the time point separating the acceleration and deceleration phases of the PMNL oxygenation response to stimulation and as such satisfies the criterion of a selected condition of PMNL activation.

Polyvalent antisera to Pseudomonas ribosomal vaccines: protection of mice against clinically isolated strains.

The preparation of polyvalent antisera to ribosomal vaccines from Pseudomonas aeruginosa is described. The ability of these antisera to protect mice by passive immunization against challenge with randomly chosen, clinically isolated strains of P. aeruginosa is reported. Significant protection was achieved against 34 of 40 strains tested (85%). Included among these strains against which protection was achieved were four mucoid strains. In addition, the degree of cross-protection attainable by the ribosomal vaccines was investigated. The results obtained indicated that these vaccines are generally serotype specific.

Passive immunization against Pseudomonas with a ribosomal vaccine-induced immune serum and immunoglobulin fractions.

Passive protection of mice against Pseudomonas aeruginosa using specific antisera and immunoglobulin fractions induced by immunizing rabbits with a ribosomal vaccine is reported. The results demonstrated that protection by the ribosomal vaccine against challenge with live organisms can be serum mediated. Previous work has shown that the vaccine can be separated into two components on the basis of molecular weight and that both higher (peak A)- and lower (peak B)-molecular-weight fractions were capable of inducing active immunity in mice. The present report indicates that both fractions are also capable of eliciting the production of mouse-protective antibody in rabbits. Agar gel diffusion with antisera to peaks A and B or unfractionated vaccine indicated a common antigenic component among them in addition to an extra antigen in unfractionated vaccine not present in peak B. Passive hemagglutination with antisera to peaks A and B demonstrated high-titer agglutinating antibody only with antiserum to peak A when a method of erythrocyte sensitization for lipopolysaccharide antigens was used. Also, passive hemagglutination was greatly inhibited by small amounts of lipopolysaccharide prepared from the same organism from which the vaccine was made. Both antisera to peaks A and B fixed complement with either A or B antigens. Antisera to peaks A and B, when reacted with peak B antigen, had about the same complement fixation titer (as determined by a quantitative complement fixation test). However, when peak A antigen was used, antiserum to peak A had about twice the complement fixation titer that antiserum to peak B had. These results are consistent with previous observations which suggest that the ribosomal vaccine contains lipopolysaccharide in addition to an unidentified immunogenic principle associated with ribosomes. Furthermore, this immunogen was present in both peaks A and B, but detectable amounts of lipopolysaccharide were present only in peak A. The relative importance of the immunoglobulin G (IgG) and IgM classes of antibodies was also compared. The results indicated that both IgG and IgM isolated from immune rabbit serum are protective in mice. Only IgG precipitated with the vaccine in agar gel diffusion, but both IgG and IgM were active in passive hemagglutination and in complement fixation. The passive hemagglutination titer of the IgM was higher than that of the IgG, but the complement fixation titer of the IgG was higher than that of the IgM. The mouse-protective capability of the IgG and IgM was about the same.

Pharmacokinetics of naloxone and naltrexone in the dog.

The serum kinetics of 5 mg/kg of i. v. naloxone or naltrexone were studied in each of two groups of five dogs; serum samples were obtained from 2 min to 2 hr after injection. Serum concentrations were determined by radioimmunoassay. Serum levels of both naloxone and naltrexone fell rapidly; serum half-life during the elimination phase was 71.2 +/- 8.9 min (mean +/- S.E.) for naloxone and 85.1 +/- 9.0 min (mean +/- S.E.) for naltrexone. Although human and dog kinetics are similar for naloxone, naltrexone is long-acting in man, but is quickly dissipated in the dog.

Pseudomonas ribosomal vaccines: preparation, properties, and immunogenicity.

The preparation, properties, and immunogenicity of ribosomal vaccines from Pseudomonas aeruginosa are described. These preparations, containing protein and RNA, were tested for immunogenicity by active immunization of mice and subsequent challenge with homologous, live bacteria. The results demonstrated that vaccines prepared from a majority of serotypes used were immunogenic, i.e., afforded 60 to 100% mouse protection against a challenge inoculum containing 8 to 50 50% lethal doses. In some cases vaccine doses as low as 1 microgram of RNA provided 100% mouse protection. Molecular sieve chromatography of a highly immunogenic ribosomal preparation on Sepharose 4B demonstrated the presence of two molecular weight fractions: (i) peak A, an excluded peak (thus having a molecular weight of at least 2 times 10(7)), and (ii) peak B, considerably retarded, with an elution position corresponding to a molecular weight of about 2.2 X 10(6), approximating that of typical 70S ribosomes. Both peaks A and B were immunogenic; however, the immunogenicity of peak A was greater (i.e., a smaller immunizing dose was required) than that of peak B. Peak A was shown to contain components of lipopolysaccharide in addition to protein and RNA (which comprised 80% of the dry weight of peak A). On the other hand, peak B was shown to be free of lipopolysaccharide, and 100% of its dry weight consisted of protein and RNA.