Leukopenic Mice Research Articles

A protective human monoclonal antibody directed to the outer core region of Pseudomonas aeruginosa lipopolysaccharide

The protective activity against experimental Pseudomonas aeruginosa infection of a human monoclonal antibody, MH-4H7, which is thought to recognize L-rhamnose and its neighboring residues in the outer core region of P. aeruginosa lipopolysaccharide and which binds to strains of Homma serotypes A, F, G, H, K, and M, was studied in normal, burned, and leukopenic mice. MH-4H7 at doses of 0.1 to 1.0 micrograms per mouse (5 to 50 micrograms/kg) was effective against serotype A, F, G, H, and K clinical isolates of P. aeruginosa tested in normal mice but not against strains of serotype M, B, E, or I. The 50% protective doses were calculated to be 0.01 and 0.1 micrograms per mouse against challenge with serotype G strains and 3 to 8 micrograms per mouse against challenge with serotype A strains. MH-4H7 promoted macrophage-mediated opsonophagocytosis of serotype A, F, G, H, and K strains but not of serotype M strains. The opsonophagocytic activity, expressed as the reduction rate of viable bacteria in the presence of MH-4H7, macrophages, and complement, was higher against serotype G strains (more than 90%) than against serotype A strains (60 to 80%) and serotype F, H, and K strains (50 to 86%). It was correlated with the protective activity but not with the binding intensity of MH-4H7 to the organisms. In addition, burned and leukopenic mice as well as normal mice infected with serotype G strains recovered from a very low dosage of MH-4H7. Thus, a monoclonal antibody directed to the outer core region of P. aeruginosa lipopolysaccharide was effective against infection with a wide range of O-serotype strains of P. aeruginosa.

Comparative Therapy with Cefpirome Alone and in Combination with Rifampin and/or Gentamicin against a Disseminated Pseudomonas aeruginosa Infection in Leukopenic Mice

Treatment of disseminated Pseudomonas aeruginosa infection in leukopenic mice was evaluated using cefpirome alone and in combination with gentamicin and/or rifampin. Mice were made leukopenic with cyclophosphamide and infected through a skin incision with an inoculum of 1250 organisms (13 LD50). Antibiotics were administered subcutaneously for 48 h. Although the addition of cefpirome to gentamicin and/or rifampin improved survival significantly at 48 h compared with untreated controls (84.6%-100% vs. 38.5%), therapy with these combinations did not improve survival significantly from that achieved with cefpirome alone. Quantitative blood and tissue (liver, spleen, kidney, lung) cultures in mice treated with cefpirome alone or including rifampin were lower than in infected controls or groups receiving therapy that excluded cefpirome. Highest counts were observed in mice receiving cefpirome plus gentamicin. Except for the cefpirome plus gentamicin group, which demonstrated areas of acute tubular necrosis, the cefpirome group had less tissue pathology than infected controls.

In vitro and in vivo activities of Sch 39304, fluconazole, and amphotericin B against Histoplasma capsulatum

The antifungal activities of amphotericin B and two triazoles, Sch 39304 and fluconazole, were tested against Histoplasma capsulatum. In this study Sch 39304 compared favorably with amphotericin B in treating histoplasmosis in normal and leukopenic mice, whereas fluconazole was much less active. The differences in the efficacies of the triazoles appeared to be due to differences in their pharmacokinetics and the dosage schedule that was used. For amphotericin B there was a good correlation between in vitro and in vivo efficacy, but this was not true of the triazole derivatives. These results further demonstrate that, with the methods used in this study, in vitro susceptibility testing of triazoles may not be predictive of in vivo activity against isolates of H. capsulatum.

Comparison of Fluconazole with Amphotericin B in Treatment of Histoplasmosis in Normal and Immunosuppressed Mice

The activities of fluconazole and amphotericin B against Histoplasma capsulatum were investigated. The minimum inhibitory concentrations ranged from 0.12 to 0.47 microgram/mL for amphotericin B and from 16 to 250 micrograms/mL for fluconazole. Fluconazole given orally twice a day for 6 consecutive days compared favorably with amphotericin B given intraperitoneally once every other day for a total of six doses in the treatment of histoplasmosis in normal and leukopenic mice.

Interleukin-1 alpha: its possible roles in cancer therapy.

Our studies on recombinant human IL-1 alpha polypeptide were summarized with respect to molecular cloning, production, quantitative assay systems, antitumor activity, myelorestorative activity and augmentation of host resistance to infections. Recombinant human IL-1 alpha (18 kDa) was produced through the expression of the cloned human IL-1 alpha cDNA in Escherichia coli and purified to an endotoxin-free homogeneous polypeptide. The human IL-1 alpha inhibited dose-dependently the growth of syngeneic murine tumors transplanted in mice and completely regressed the tumors in some cases, and its antitumor activity was significantly enhanced in combination with indomethacin. The human IL-1 alpha accelerated the recovery of the numbers of peripheral leukocytes and neutrophils in a dose-dependent manner at a dose as low as 10 ng/mouse/day in myelosuppressed mouse model produced by administering anticancer chemotherapeutic drugs. The myelorestorative effect of IL-1 alpha was observed not only on leukocytes/neutrophils, but also on platelets in myelosuppressed mice. In addition, the human IL-1 alpha markedly augmented dose-dependently resistance of normal and leukopenic mice to various microbial infections. These results suggested that recombinant human IL-1 alpha might be useful for cancer therapy from the viewpoints of improving adverse effects such as myelosuppression caused by chemotherapy and/or radiation therapy and preventing infections. In addition, use of IL-1 alpha may permit more intensive chemo- and radiation therapies using higher doses. Finally, the antitumor activity of the IL-1 alpha itself may play an important role.

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 factortreated) 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.

COMPARISON OF ORAL AND PARENTERAL GENTAMICIN (G) IN THE PREVENTION OF ENTERALLY-ACQUIRED PSEUDOMONAS (PS) SEPSIS

We evaluated the protective effect of oral and parenteral G in the leukopenic mouse. C3H/HeN mice were made leukopenic by injection of cyclophosphamide. Mice were either gavage fed (po) or injected (ip) with G in 5% glucose water (D5W) at 5, 10, or 15 mg/kg/day (days 1-7). On day 4 the animals were orogastrically inoculated with Ps. aeruginosa, immunotype 7 (ATCC #27318) in D5W. Survival of treatment groups were compared by 2-way ANOVA.RESULTS:The MIC of G for PS is 2 μg/ml. Protection by G is significantly related to dose [F(2,55)=5.65, p=0.0059]. Prophylactic G is more protective at 15 mg/kg/day than 5 mg/kg/day (p<0.05). Interestingly, oral G is as protective as parenteral G when given at equivalent doses.CONCLUSION: Prophylactic oral and parenteral G are effective in the prevention of enterally-acquired Ps sepsis in a leukopenic mouse model.

1007 ORAL ANTI-PSEUUOMONAS (Ps) IgG PROVIDE SPECIFIC IMMUNE PROTECTION AGAINST PS SEPSIS DURING CYCLOPHOSPHAMIDE-INDUCED LEUKOPENIA

We previously showed that orally-administered bovine IgG antibodies resist proteolysis and provide specific immune protection. In this report we use specific anti-Ps bovine IgG to reduce the mortality of Ps sepsis in an immunocompromised host. We used a leukopenic mouse model to evaluate the protective effect of orally-administered anti-Ps bovine colostral (BCI) and human serum (HISG) IgG and three other antimicrobial factors - lacto-ferrin (LF), lysozyme (LZ) and lactoperoxidase (LZ). Leukopenic mice (cyclophosphamide, 300 mg/kg) were fed anti-Ps BCI, control BCI, commercial HISG, LF, LZ, LP, or a 57. glucose solution (D5W) over a 6-day period. Anti-Ps BCI, control BCI, and HISG had anti-Ps activity by ELISA, agglutination, and complement fixation assays. On day 4 of feedings, the mice were orally-inoculated with 106 Ps. aeruginosa (serotype 10). Mortality was recorded 24, 48 and 72 hours later. Group survival curves were not equal (p<0.01; Kruskal-Wallis). Paired comparison tests for the anti-Ps BCI and D5W groups showed a decrease in mortality of ≍50% (p<0.05). Groups fed control BCI or HISG, which exhibit less anti-Ps activity, had intermediate reductions in mortality. The mortality of groups fed LF, LZ, or LP was not different from the D5W group. We conclude that oral anti-Ps BCI reduces the mortality of Ps sepsis in a murine model of leukopenia. We speculate that oral antibodies to enteric organisms may be an effective means of reducing the incidence and severity of gram negative sepsis in leukopenic hosts.

Prophylaxis of Pseudomonas aeruginosa infections in leukopenic mice by a combination of active and passive immunization.

Mice rendered leukopenic with cyclophosphamide and then challenged with viable Pseudomonas aeruginosa were used to determine the protective efficacy of active immunization against exotoxin A and of passive immunization with human antiserum to Escherichia coli J5, a rough mutant of Escherichia coli O111:B4. Neither treatment alone provided a greater degree of protection than its respective control. However, the combination of these treatments produced a moderate, yet consistent, increase in the survival of infected immunosuppressed mice.

ORALLY-ADMINISTERED BOVINE COLOSTRAL IMMUNOGLOBULINS (BCI) PROTECT LEUKOPENIC MICE AGAINST Ps. AERUGINOSA (Ps)

We immunized pregnant cows with Ps Extract Vaccine (Wellcome, UK) to produce anti-Ps BCI. Anti-Ps BCI have agglutinating and opsonizing activity against Ps, but are not bacteriacidal or static. By adsorption with Ps, these activities can be removed from BCI. Bovine IgGl can be identified on the surface of Ps adsorbed with anti-Ps BCI. We compared the protective effect of orally-fed anti-Ps BCI, control BCI from non-immunized (NI) cows, pooled human immune globulin (HIG), and D5W on the mortality of leukopenic mice orally-inoculated with Ps. Mice (18-22g) were made leukopenic with cyclophosphamide, fed globulins or D5W daily (days 1-6) per gavage, and orally-inoculated on day 4 with 106 Ps serotype 10. WBCs on day 4 were 2331 ± 696. Survival data were evaluated by the Kruskal-Wallis statistic and nonparametric log-rank test with adjustment for multiple comparisons.Treatment group survival curves were not equal (p<0.01). Anti-Ps BCI-fed animals had less mortality than NI-BCI, HIG, or D5W-fed animals, but these differences reached statistical significance only for anti-Ps BCI vs D5W (z=3.676, p<0.05). Conclusion: Orally fed anti-Ps BCI may reduce the incidence and mortality of Ps sepsis in the transiently leukopenic host

Importance of extracellular and cell-bound beta-lactamase in mediating resistance of Staphylococcus aureus to mezlocillin.

Most penicillin-resistant staphylococci release a considerable portion of their beta-lactamase into the surrounding medium. Accumulation of this exoenzyme in conventional susceptibility test systems may result in a rapid inactivation of hydrolyzable antibiotics. Since under in vivo conditions the concentration of extracellular beta-lactamase should depend on the site of infection, susceptibility of Staphylococcus aureus to mezlocillin, a broad-spectrum penicillin, was measured in an open test model which prevented build-up of exoenzyme. The staphylococcal cells were immobilized and incubated between two membrane filters, and the excreted beta-lactamase was washed out by a constant flow of broth containing the antibiotic. Two test strains which produced large amounts of extracellular beta-lactamase and which were found to be resistant in the broth dilution test proved to be susceptible to mezlocillin in the open test model. This indicates that resistance to mezlocillin as measured by the broth dilution method was mediated predominantly by the extracellular enzyme fraction. Experiments performed with small infective doses in a model of peritoneal infection in leukopenic mice suggest that mezlocillin exhibits a therapeutic effect against beta-lactamase-producing staphylococci under certain in vivo conditions in which build-up of extracellular beta-lactamase does not occur.

Passive protection against Pseudomonas aeruginosa infection in an experimental leukopenic mouse model.

An experimental leukopenic mouse model was used to evaluate the protective capacities of immunoglobulin G (IgG) fractions directed against toxin A (AT-IgG), elastase (AE-IgG), and lipopolysaccharide (ALPS-IgG) against fatal Pseudomonas aeruginosa infection. Statistically significant protection, as measured by long-term survival, was observed only when mice were treated with serotype-specific ALPS-IgG. The mean lethal dose for P. aeruginosa could be increased by as much as 6,600-fold for mice given ALPS-IgG as compared to mice which received only normal rabbit IgG. ALPS-IgG afforded high levels of protection, even when administered up to 6 h postchallenge. Experiments designed to monitor the growth and spread of a locally administered challenge showed that ALPS-IgG prevented bacteremia and organ colonization, which were pronounced in control animals. The effectiveness of combined antibiotic and immune therapy was tested. Gentamicin alone or in combination with AT-IgG or AE-IgG provided no detectable protection. However, its use with ALPS-IgG afforded substantially higher levels of protection than ALPS-IgG alone.

Simple model for the study of Pseudomonas aeruginosa infections in leukopenic mice.

A simple, reproducible model of fatal Pseudomonas aeruginosa sepsis in mice during immunosuppression was developed. Mice were rendered leukopenic (less than or equal to 800 leukocytes per mm3 of blood) for a period of 5 days by multiple injections of cyclophosphamide. Mice were challenged at the onset of leukopenia by instilling the bacteria onto a 0.5-mm incision made into the back. The mean lethal dose (LD50) for P. aeruginosa PA220 and M-2 was less than 20 bacteria. The mean time to death for these strains ranged from 46 to 59 h. Leukopenic mice were comparatively resistant when challenged with Klebsiella pneumoniae (LD50 = 1.5 x 10(6)) or Staphylococcus aureus (LD50 greater than 10(6)). Infection with P. aeruginosa was characterized by rapid bacterial multiplication in the skin at the site of infection, producing ecthyma gangrenosum. Bacteremia and colonization of the liver were pronounced 21 h postinfection. This model should prove to be a useful tool for studying the pathogenesis of P. aeruginosa infections under immunosuppressed conditions.

Selection of aminoglycoside-resistant variants of Pseudomonas aeruginosa in an in vivo model.

The therapeutic failure of aminoglycosides in leukopenic mice was studied. Pseudomonas aeruginosa isolated from experimentally infected leukopenic mice was analyzed for susceptibility to gentamicin. Bacterial population analyses were performed before, during, and after therapy with high doses of gentamicin. Inocula from numerous strains, including P. aeruginosa strain ATCC 27853, harbored slowly growing aminoglycoside-resistant subpopulations. In leukopenic mice (in contrast to normal mice) receiving gentamicin, the breakthrough growth of these subpopulations occurred 6 hr after treatment. Resistance to gentamicin reversed in vivo when gentamicin treatment was stopped. The emergence of gentamicin-resistant subpopulations was successfully suppressed by a combined treatment regimen of gentamicin and ticarcillin. Gentamicin-ticarcillin synergy in these infections is due, in part, to antimicrobial suppression of slowly growing aminoglycoside-resistant subpopulations of bacteria.

Experimental nasal infection of normal and leukopenic mice with Pseudomonas aeruginosa.

Histological and ultrastructural changes in the nasal mucosa of normal and leukopenic mice exposed to Pseudomonas aeruginosa were compared and correlated with changes in the distribution of pseudomonads by use of immunoperoxidase labeling. Pseudomonas was limited to the surface of the nasal mucosa of normal mice and was cleared rapidly. Concurrently, granulocytes were recruited across unaltered nasal epithelium and contained phagocytosed bacilli within two hours. Pseudomonas was limited to the surface of the nasal mucosa of most leukopenic mice at two hours. By four hours, pseudomonads had penetrated interepithelial junctions of all leukopenic mice. Granulocytes were not recruited and nasal epithelium underwent necrosis at points of invasion. These results show that neutrophils participate in the clearance of P. aeruginosa from the surface of the nasal mucosa and that the failure to recruit granulocytes may be important in the breakdown of epithelial barriers. Possible mechanisms of mucosal invasion are discussed.