Tumor models: assessing toxicity in efficacy studies.

Efficacy studies in animal tumor models provide an early opportunity to collect preliminary information on toxicity. When screening and evaluating cytotoxic chemotherapeutic agents, efficacy studies usually include at least 1 dosage level that causes severe toxicity and death. Pathologic evaluation in early efficacy studies may reveal major target organs, dosage/schedule relationships, pharmacokinetic/toxicity relationships, effects of formulation and route of administration, maximum tolerated dose, cause of death, and reversibility of changes in normal tissues. Intraperitoneal formulations are frequently used to establish proof of concept for promising compounds (hits) from in vitro screens; however, these crude formulations may also induce intraperitoneal inflammation and confound the interpretation of both efficacy and toxicity. Efficacy studies conducted in the later stages of drug discovery may be used to refine the dose and schedule proposed for phase I clinical trials. Efficacy studies in animal tumor models provide useful toxicologic data for screening potential drug candidates, optimizing the therapeutic index, and designing both preclinical and clinical development programs.

Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

Effect of passively administered immunoglobulin G on the colonization and clearance of Bordetella avium in turkeys.

This study was conducted to detect the effect of parenterally administered immunoglobulin isotype G (IgG) on the colonization and clearance of Bordetella avium at the tracheal surface in young turkeys. In two separate experiments, 3-week-old turkeys were infected with B. avium either after or before IgG administration. Comparisons were made between a control group which received an irrelevant IgG (specific for keyhole limpet hemocyanin [KLH]) and the experimental group which received a B. avium-specific IgG. When given before the bacteria, IgG reduced the numbers of colony-forming units (CFUs) in the trachea. As a supplement to non-specific respiratory defense mechanisms, B. avium-specific IgG also appears to inhibit colonization of the tracheal mucosa. In a second experiment designed to study the role of IgG in bacterial clearance, administration of B. avium-specific IgG after bacterial inoculation significantly reduced the number of CFUs on the tracheal surface. These studies support the role of B. avium-specific IgG in resistance to and recovery from B. avium infection.

A time-course study of the transfer of immunoglobulin G from blood to tracheal and lacrimal secretions in young turkeys.

Three-week-old turkeys were injected intravenously with Bordetella avium-specific immunoglobulin G (IgG), and absorbance readings were measured in blood, tracheal washings, and lacrimal secretions using an enzyme-linked immunosorbent assay at various time intervals. IgG was detected in tracheal and lacrimal secretions as early as 5 minutes after injection and peaked at 10 minutes after injection. Thereafter, IgG absorbance declined rapidly, reaching background levels by 24 hours. The absorbance readings of IgG in all three sites were comparable at all times from 10 minutes to 24 hours after administration. The results indicated that movement of IgG from blood to mucosal surfaces in turkeys occurs rapidly.

Canine distemper epizootic in lions, tigers, and leopards in North America.

Canine distemper virus (CDV) infection occurred in captive leopards (Panthera pardus), tigers (Panthera tigris), lions (Panthera leo), and a jaguar (Panthera onca) in 1991 and 1992. An epizootic affected all 4 types of cats at the Wildlife Waystation, San Fernando, California, with 17 mortalities. CDV-infected raccoons were thought to be the source of infection in these cats. Two black leopards died at the Naibi Zoo, Coal Valley, Illinois, and 2 tigers died at the Shambala Preserve, Acton, California. Initial clinical signs were anorexia with gastrointestinal and/or respiratory disease followed by seizures. Canine distemper virus was isolated from 3 leopards, 3 tigers, and 3 lions that died or were euthanized when moribund. Monoclonal antibody testing identified the virus isolates as CDV. Gross and histopathologic findings were similar to those found in canids with distemper with a few exceptions. There were fewer lesions in the brain, and there was a pronounced type 2 cell proliferation in the lung, with inclusion bodies and CDV antigen demonstrated by immunohistology. Neutralizing antibody to CDV was found in high titers in serum from most animals but was absent or was found only in low titers in some cats that succumbed after CDV infection. There was a marked difference in neutralizing antibody titers when tests were done with different strains of CDV.

Mucosal and systemic humoral immune response to Bordetella avium in experimentally infected turkeys.

Antibody response to Bordetella avium was measured in serum and mucosal secretions of experimentally infected turkeys. Two-day-old turkeys were inoculated with B. avium, and four inoculated turkeys and four uninoculated control birds per trial were euthanatized weekly from 1 through 8 weeks postinoculation (PI). Maternal antibody of the IgG isotype, present in all 2-day-old birds sampled, decreased to background levels by 3 weeks of age. Antibody (IgG, IgM, IgA) was detected in serum, tracheal washings, and lacrimal secretions in response to B. avium infection. Regardless of the sample site and isotype, antibody levels peaked at 4-6 weeks PI and then decreased rapidly from 6 to 8 weeks PI. In general, IgM and IgA levels peaked earlier (4-5 weeks PI) but declined more rapidly than IgG levels. Numbers of B. avium in the trachea peaked at 2-3 weeks PI and then decreased rapidly from 4 to 8 weeks PI. Even though no direct causal relationship could be determined, the results indicate that an increasing level of antibody in serum, tracheal washings, and lacrimal secretions is temporally associated with clearance of B. avium from the trachea.

Uptake of ferritin and Bordetella avium in bronchus-associated lymphoid tissue of turkeys.

The uptake of macromolecular and particulate materials in bronchus-associated lymphoid tissue (BALT) in turkeys was examined using transmission electron microscopy. Tracer materials used were live and ultraviolet-killed (UV-killed) Bordetella avium and ferritin. Suspensions of bacteria and ferritin were instilled via intratracheal catheterization and allowed to remain in contact with the respiratory surfaces for 0, 10, 30, 60, 90, and 120 min. Ferritin and B. avium were taken up by both ciliated and non-ciliated cells of the epithelium overlying BALT (BALT epithelium). Ferritin was found in organelles associated with endocytosis (i.e. apical vesicles, endosomes, cytoplasmic vacuoles) and was apparently transported across epithelial cells, since it was also found in intercellular spaces. Bacteria were found in vacuoles within BALT epithelial cells, but not free in intercellular spaces. Some macrophages in BALT epithelium also contained bacteria. No differences were observed between uptake of live and UV-killed bacteria. We conclude that both ciliated and non-ciliated cells of BALT epithelium in turkeys are able to take up macromolecular and particulate materials. Bacteria are also accessible to intraepithelial macrophages, although whether they are taken up directly from the bronchial surface or whether they pass through epithelial cells first could not be determined. This evidence suggests that antigens, including respiratory pathogens, could gain access to cells of the avian immune system by transepithelial passage in BALT.

Glycoconjugates as components of receptors for Bordetella avium on the tracheal mucosa of turkeys.

Bordetella avium is an important respiratory tract pathogen of turkeys. In common with other pathogenic bordetellae, B avium manifests a tissue tropism for cilia of the respiratory tract epithelium. To determine the molecular characteristics of the host cell receptors for B avium, we used hemagglutination and in vivo adherence assays. Carbohydrates, mucus, sialic acid-specific lectin, and other glycoconjugates were evaluated for their ability to competitively inhibit binding of B avium to host cells. The gangliosides, GD1a and GT1b, completely inhibited hemagglutination, whereas N-acetylneuraminic acid (sialic acid) partially inhibited hemagglutination. Adherence to turkey tracheal mucosa in vivo was significantly (P < 0.01) inhibited by GD1a and GT1b gangliosides, N-acetylneuraminic acid, bovine submaxillary mucin, and horseshoe crab (Limulus polyphemus) lectin. Treatment of the tracheal mucosa with neuraminidase also inhibited adherence of B avium. We conclude that N-acetylneuraminic acid and the gangliosides, GD1a and GT1b, may be important components of the tracheal mucosa receptor for B avium in turkeys.

A monoclonal antibody-based latex bead agglutination test for the detection of Bordetella avium.

The purpose of this study was to develop a rapid method to distinguish Bordetella avium from closely related Bordetella avium-like and B. bronchiseptica bacteria. A monoclonal antibody of the IgM isotype was produced in Balb/c mice against live B. avium strain 75. The monoclonal antibody, in the form of ascites fluid, was added to a bovine serum albumin-glycine buffer (pH 8.6) and adsorbed to 3.03-microns-diameter latex beads. Optimum concentrations of antibody, beads, and bacteria were determined. The latex bead conjugate was tested against 40 isolates of B. avium, 24 isolates of B. avium-like bacteria, 17 isolates of B. bronchiseptica, two isolates of Alcaligenes faecalis, and several other common genera. Strong agglutination occurred with all B. avium isolates and the two isolates of A. faecalis. Weak agglutination occurred with Staphylococcus aureus and two isolates of B. bronchiseptica. There was no agglutination with any of the B. avium-like isolates. The latex bead agglutination test may be useful as an aid in the identification of B. avium when used in conjunction with other criteria.

Distribution and quantitation of plasma cells, T lymphocyte subsets, and B lymphocytes in bronchus-associated lymphoid tissue of chickens: age-related differences.

Development of the lymphoid cell compartment in bronchus-associated lymphoid tissue (BALT) of specific pathogen free chickens was examined. Specifically, B lymphocytes, T cell subsets (CD4 and CD8), and IgA-, IgG-, and IgM-producing plasma cells were labeled using immunocytochemical methods. Immunoglobulin-producing cells (IgPC) were quantitated, and comparisons of IgPC numbers were made among chickens of different ages, among immunoglobulin isotypes, and between lymphoid (BALT) and nonlymphoid (non-BALT) areas in the primary bronchus. At hatching, BALT was devoid of IgPC, but by 2 weeks of age cells producing IgA, IgG, and IgM were present. Initially, there were approximately equal numbers of IgA-, IgG-, and IgM-PC; after 2 weeks of age, however, IgG- and IgM-PC outnumbered IgA-PC. At all ages, IgPC were more numerous in non-BALT regions of the primary bronchus than in BALT regions. Small numbers of T and B lymphocytes were present in BALT from 1-day old chickens, but substantial populations of these cells were not seen until 1-2 weeks of age. T helper (CD4+) cells were found near B cell regions in BALT lymphoid nodules, while T cytotoxic/suppressor (CD8+) cells were more evenly distributed throughout the nodules and in the epithelium. B lymphocytes predominated in germinal centers and also overlapped CD4+ populations adjacent to germinal centers. Lymphocyte cell types needed to initiate and regulate immune responses are present in chicken BALT and may be involved in protecting poultry from respiratory pathogens.

Structure and development of bronchus-associated lymphoid tissue in conventionally reared broiler chickens.

The development of bronchus-associated lymphoid tissue (BALT) in conventionally reared broiler chickens of 1 day and 1, 2, 3, 4, 6, and 8 weeks of age was studied using light and electron microscopy (scanning and transmission). BALT in these chickens resembled other mucosa-associated lymphoid tissues (MALT) in that it was composed of an altered epithelium overlying a population of lymphocytes and contained potential antigen-presenting cells, such as macrophages and dendritic cells; high endothelial venules were also present. In contrast to other MALT, epithelial cells in chicken BALT were not of the M-cell type; i.e., they lacked large numbers of apical tubules and vesicles for specialized uptake of luminal antigens. There were age-related differences in size, number, and cellular composition of BALT nodules. Lymphoid nodules were progressively larger and more numerous with increasing age. Germinal centers were present in birds 2 weeks of age and older. BALT epithelium was primarily squamous and non-ciliated in 1-day and 1-week-old chicks, becoming progressively more columnar and ciliated in older chickens. Lymphocyte infiltration of the epithelium was extensive at 1 to 4 weeks of age; in older chickens, distinct epithelial and lymphocytic compartments were separated by connective tissue.

Colonization of the small intestine of weaned pigs by enterotoxigenic Escherichia coli that lack known colonization factors.

Intestinal colonization of 3-week-old weaned pigs by enterotoxigenic Escherichia coli (ETEC) strains that were originally isolated from weaned pigs with fatal diarrhea and that lacked K88, K99, F41, and 987P adhesins (4P- ETEC) was studied by histologic, immunofluorescent, and electron microscopic techniques. In the first experiment, 16 principal pigs were inoculated orogastrically with ETEC strain 2134 (serogroup O157: H19) or 2171 (serogroup 0141:H4), and eight control pigs were not inoculated. In the second experiment, 24 principals were inoculated with ETEC strain 2134, and 12 controls were inoculated with a nonenterotoxigenic strain of E. coli. Principal and control pigs were necropsied at intervals from 24 to 72 hours after inoculation of principals to provide the tissues used for this report. Results from the two experiments and with both ETEC strains were similar and therefore were combined. Adhesion by 4P- ETEC was demonstrated in ileum but not in cecum or colon in 22/40 principal pigs sampled at 24 to 72 hours after orogastric inoculation. Adherent bacteria were most apparent on the intestinal villi covering Peyer's patches. Only occasional adherent bacteria were detected in ileal sections from a few (4/20) of the control pigs. Adherence by 4P- ETEC was characterized by "patches" of bacteria closely associated with the lateral surfaces and less frequently with the tips and the bases of intact villi. In most cases, the adherent bacteria were separated from epithelial cell microvilli and other bacterial cells by a 50-400-nm space. Filamentous bacterial appendages bridged this space and formed a network among adjacent bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental model for subclinical edema disease (Escherichia coli enterotoxemia) manifest as vascular necrosis in pigs.

An experimental model for subclinical edema disease was developed in weanling pigs. In multiple experiments, 3-week-old pigs were weaned, then inoculated intragastrically with 10(10) colony-forming units of an SLT-IIv-positive strain of Escherichia coli originally isolated from a pig with edema disease (principals). Control pigs were inoculated with a nonpathogenic E coli strain. Of 39 principals, 8 developed clinical edema disease within 14 days after inoculation. However, 20 of 21 principals that did not develop clinical signs of edema disease, but were submitted for necropsy examination at 14 days after inoculation, had characteristic vascular lesions of edema disease. Vascular lesions, found principally in ileum and brain, consisted of segmental necrosis of myocytes in the tunica media of small arteries and arterioles. None of the pigs inoculated with a nonpathogenic strain of E coli developed edema disease or vascular lesions. None of the principals necropsied at 2 days after inoculation had vascular lesions. Development of vascular lesions by 14 days after inoculation was used as the end point for detecting subclinical edema disease in the model.

Morphologic characterization of conjunctiva-associated lymphoid tissue in chickens.

Conjunctiva-associated lymphoid tissue (CALT) in the eyelids of chickens was studied by gross, histologic, and electron microscopic techniques. Structural features were characterized at 1 day of age and at posthatching week (PHW) 1, 2, 3, 4, 6, 8, 12, and 16. Beginning at PHW 1, prominent lymphoid nodules containing a heterogenous population of lymphocytes, lymphoblasts, and macrophages were first observed within conjunctival folds and fissures of the lower eyelid. Nodules contained germinal centers by PHW 2 and plasma cells by PHW 4. The epithelium associated with these nodules was flat, had short, irregular microvilli, contained intraepithelial lymphocytes, and lacked goblet cells. High endothelial venules were located at the base of lymphoid nodules and contained lymphocytes within and below the cuboidal endothelium. In the upper eyelid, CALT was morphologically similar to lymphoid tissue in the lower eyelid, but nodules were smaller and more random, lacked association with epithelial folds and fissures, and were clustered around the opening of the nasolacrimal duct. By PHW 12, CALT was characterized by basal germinal centers outlined by collagenous stroma, suprafollicular plasma cells, columnar epithelium with goblet cells, and fewer intraepithelial lymphocytes. On the basis of these features, CALT in chickens has morphologic characteristics similar to other components of the mucosal immune system and, therefore, may have a role in mucosal immunity.

Particle uptake by conjunctiva-associated lymphoid tissue (CALT) in turkeys.

Uptake of tracer particles was assessed in lower eyelid conjunctiva-associated lymphoid tissue (CALT) of 3-week-old turkeys. Tracer particle suspensions, including carbon, iron oxide, or three sizes of latex beads (0.81 micron, 1.7 microns, and 2.9 microns), were placed into the experimentally sealed conjunctival space. After 5, 15, or 30 minutes, eyelids were removed and CALT was examined by light microscopy. Uptake was confirmed for all tracers and occurred within the lymphoepithelium of CALT. The uptake of latex beads was not as frequent as for carbon and iron. Tracers were increasingly evident in lymphoepithelium and subepithelial macrophage clusters as contact time increased. These findings provide additional evidence that CALT is capable of antigen uptake and may play a role in paraocular and upper respiratory immunity in turkeys.

Quantification of particle uptake by conjunctiva-associated lymphoid tissue (CALT) in chickens.

Tracer particle uptake by conjunctiva-associated lymphoid tissue (CALT) was quantified in the lower eyelids of 1-, 3-, 5-, and 7-week-old broiler chickens. CALT was measured histologically by computerized image analysis in all birds and in additional 1-day-old and 16-week-old chickens not subjected to uptake assessment. Suspensions of carbon or iron oxide were placed in contact with CALT for 5, 15, or 30 minutes (contact time). After eyelid removal, tracer uptake was scored by light microscopy, CALT was measured, and a mathematically derived uptake index was evaluated statistically. At each age examined, computer-generated measurements showed a significant increase in the proportion of CALT lymphoepithelium within proximal eyelids. Within the conjunctival sites evaluated, tracer uptake was significantly greater in lymphoepithelium than in non-lymphoepithelium at all ages and at all contact times. Uptake increased significantly between 3 and 5 weeks of age and between 5 and 15 minutes of tracer contact. Based on these uptake data for CALT in chickens, a minimal age-specific maturity is suggested that may influence function in mucosal immunity.

A morphologic study of bronchus-associated lymphoid tissue in turkeys.

Bronchus-associated lymphoid tissue (BALT) in normal turkeys of ages 1 day and 1, 2, 3, 4, 8, and 18 weeks was examined by light microscopy and by scanning and transmission electron microscopy. Turkey BALT resembled other mucosa-associated lymphoid tissues; it was made up of a population of lymphocytes covered by a specialized epithelium different from typical pseudostratified ciliated columnar bronchial epithelium. There were distinct age-related differences in BALT structure. Bronchus-associated lymphoid nodules were larger and more numerous in older turkeys. In 1-day- to 2-week-old turkeys, the primary cell type of BALT epithelium was nonciliated cuboidal; in 2-week old turkeys it was squamous; and in turkeys older than 4-weeks of age, the epithelium was primarily ciliated columnar. In 1- to 4-week old turkeys, large numbers of intraepithelial lymphocytes disrupted the normal organization of the epithelium. In older turkeys, epithelial and lymphoid cells were in discrete compartments separated by connective tissue. Lymphocytes in 1-day-old turkeys were found in loose aggregates around venules and within the epithelium. In 1-week old turkeys, lymphocytes were organized into compartments of morphologically similar cells. By 3-weeks of age, lymphocytes were present in distinct germinal centers. Epithelial cells of BALT did not have large numbers of apical vesicles and thus were not structurally specialized for antigen uptake by endocytosis. However, the epithelial barrier appeared to be disrupted over lymphoid nodules, suggesting that antigen would be readily available to lymphocytes and phagocytes in BALT. Age-related differences in turkey BALT structure may have functional consequences with respect to the respiratory immune response.

Identification of Bordetella avium antigens recognized after experimental inoculation in turkeys.

Sera and tracheal washings (TW) were used to identify antigens of Bordetella avium recognized during experimentally induced bordetellosis in young turkeys. Pooled sera and TW were examined for antibody by a microtitration agglutination test and by western immunoblotting. In addition, comparable samples collected from 1-day-old turkeys and uninoculated control turkeys also were examined. At least 8 outer membrane proteins of B avium were recognized in immunoblots of sera and TW from infected turkeys. Reactivity of TW in immunoblots was qualitatively similar but less intense, compared with reactivity of corresponding sera collected on postinoculation (PI) weeks 2, 3, and 4. Molecular weights of the major outer membrane proteins of B avium recognized by sera and TW at PI week 4 were 100,000, 97,000, 36,000, 31,000, 21,000, 18,000, 14,000, and less than 14,000. A protein with a molecular weight of 55,000 reacted nonspecifically in all samples tested. Antibody, detectable by microtitration agglutination, was in sera of 1-day-old turkeys and in sera and TW of B avium-infected turkeys during PI weeks 2 to 4.

Orally administered clonidine as a secretagogue of growth hormone and as a thymotrophic agent in dogs of various ages.

The growth hormone (GH) secretagogue activity of variable dosages of clonidine (16.5, 50, 150, and 450 micrograms/kg of body weight), given orally mixed with the daily food ration, was evaluated in young and old dogs. Significant (P less than 0.05) increase in plasma GH concentration was detected at all dosages tested in young dogs and in response to all but the lowest dose tested in the old dogs fed the clonidine-containing diet. Old dogs had plasma GH concentration that exceeded that of young dogs when higher doses of clonidine were used. A clonidine (100 micrograms/kg)-supplemented diet was fed to middle-aged dogs twice daily for 30 days. Significant (P less than 0.01) increase of plasma GH concentration was observed on the first day of the feeding trial, but was undetectable by day 30. After feeding the clonidine-enhanced diet for 30 days, the effects on thymic morphology were variable, and there was no effect on plasma thymulin titer. Clonidine-fed dogs had significantly increased lymphocyte blastogenic responsiveness to mitogens, compared with that of control dogs, when evaluated as stimulation index.

Interaction of turkey complement with Escherichia coli isolated from turkeys.

The role of turkey complement in a serum bactericidal reaction was determined using serum-sensitive and serum-resistant Escherichia coli isolated from turkeys. Inactivation of complement by heating serum (56 C for 40 minutes) or by treating serum with 10 mM EDTA eliminated bactericidal activity. Serum-sensitive E coli organisms were killed by turkey serum treated with 10 mM ethylene glycol-bis-beta-(aminoethyl ether)-N,N,N',N'-tetraacetic acid and 5 mM MgCl2. Exposure of normal turkey serum to serum-sensitive or serum-resistant E coli resulted in equivalent reductions in hemolytic activity of serum. Treatment of serum-resistant E coli with antibody rendered the bacteria sensitive to bactericidal effects of normal turkey serum. Serum-sensitive E coli organisms were readily killed by an alternative complement pathway, serum-sensitive and serum-resistant E coli activated the complement system equally well, and antibody was required for complement-mediated killing of certain serum-resistant E coli organisms from turkeys.