Combination Therapy of Pseudomonas Aeruginosa Pyelonephritis in Neutropenic Mice With Human Antilipopolysaccharide Monoclonal Antibody and Cefsulodin

Mm-5347/96/1556-20943.W0 "HE JOURNAL OF UROLOGY Copyright 0 1996 by AMERICAN UROLOGICAL A~SOCIATION, INC.

Vol. 155,2094-2097,June 1996 Printed in U S . A

COMBINATION THERAPY OF PSEUDOMONAS AERUGINOSA PYELONEPHRITIS IN NEUTROPENIC MICE WITH HUMAN ANTILIPOPOLYSACCHARIDE MONOCLONAL ANTIBODY AND CEFSULODIN KEI ISHIBASHI*, OSAMU YAMAGUCHI, YASUO SHIRAIWA, MASAHIKO OGIHARA SHIRO SHIGETA

AND

From the Departments of Urology and Microbiology, Fukushima Medical College, Fukushima, Japan

ABSTRACT

Purpose: These studies were designed to determine the combined inhibitory effect of a human monoclonal antibody (MAb) and cefsulodin on Pseudomonas aeruginosa renal infection in a neutropenic condition. Materials and Methods: Protection against the infection of mice was estimated by survival rate and bacterial numbers in the kidney and blood. Opsonophagocytic assay by human polymorphonuclear neutrophils (PMNs) and fluorescence activated cell sorter (FACS) analysis were also examined. Results: Treatment of infected mice with MAb combined with a suboptimal dose of cefsulodin prevented the mice from developing pyelonephritis and bacteremia and resulted in a significantly higher survival rate than treatment with either MAb or cefsulodin alone (p cO.01). When bacteria were preexposed to cefsulodin, a significant enhancement in opsonophagocytic killing with MAb was observed. Fluorescence activated cell sorter analysis suggested that the bacteria incubated with l/4 minimal inhibitory concentration (MIC) of cefsulodin showed greater binding of MAb to bacteria than the control. Conclusion: The combination therapy with human antilipopolysaccharide MAb and cefsulodin is useful for P. aeruginosa pyelonephritis in neutropenic hosts. KEY WORDS:antibodies, monoclonal; pyelonephritis;cefsulodin; pseudomonas aeruginosa

Pseudomonas (P.)aeruginosa is one of the major pathogens in opportunistic bacterial infections. It frequently causes bacteremia in immunocompromised hosts, especially in patients undergoing chemotherapy or immunosuppressive therapy, as well as in those suffering from burns or cancer. Thus, in these patients, bacteremia induced by P. aeruginosa always shows a high mortality. Recent studies have revealed the possibility that passive immune therapy using a monoclonal antibody (MAb)specific for the lipopolysaccharide 0 side chain, which shows serotype-specificopsonic activity, may be useful in the treatment of P. aeruginosa infections.'" However, some investigators have indicated that treatment of P. aeruginosa infection with MAb alone was insufficient in neutropenic h o ~ t s . Since ~ . ~ the opsonophagocytic activity of polymorphonuclear cells (PMNs) is decreased considerably, some modality that effectively enhances this activity would be important in such situations. Therefore, combination therapy with MAb and an antimicrobial agent is considered one possibility for the treatment of P. aeruginosa infection. In this study, which focuses on pyelonephritis in immunocompromised hosts, we evaluated the combined inhibitory effects of MAb V9-1 and cefsulodin on P. aeruginosa pyelonephritis in neutropenic mice. In addition, its therapeutic mechanism by in vitro opsonophagocytosisof PMNs was also studied. MATERIALS AND METHODS

Reagent and buffers. Hanks' balanced salt solution containing 0.1%gelatin (GHBSS) and phosphate-buffered saline (PBS) were used for the following experiments. Cefsulodin Accepted for publication January 19, 1996. * Re uests for reprints: De artment of Urology, Fukushima Medical College, Hikarigaoka 1, fukushima 960-12, Japan.

was provided as pure chemical powder from Takeda Chemical, Osaka, Japan. Bacteria. Pseudomonas aeruginosa PA103 (Homma's serotype E) was used for all studies. The P5 strain (serotype A) was used as control. The minimal inhibitory concentration (MIC) of cefsulodin was determined by the agar dilution method in Mueller-Hinton medium. Monoclonal antibody (MAb). The production procedure of human IgM MAb V9-1 that is specific for the 0 antigen of Homma's serotype E was described by OOka et a1.6 Hybridomas producing the MAb were developed by fusion of P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethylene glycol (Mitsui Pharmaceutical, Inc., Chiba Japan). Mice. Female BALB/C mice, (8 weeks old, weighing 18 to 22 g.) were used. They were housed under sterile conditions. Urinary tract infection with P. aeruginosa in neutropenic mice. To induce a neutropenic condition in mice, cyclophosphamide was injected subcutaneously a t a dose of 250 mg./ kg. with a second dose of 100 mg./kg. on the sixth day after the first injection. The mice, which had been denied drinking water for 18 hours, were anesthetized with sodium pentobarbital. A suspension of P. aeruginosa (1.0 x lo3 CFU/ml.) in 0.05 ml. of PBS was inoculated transurethrally into the bladder, and the urethra was clamped for 4 hours. Quantitative colony counts of bacteria in the kidney and blood were carried out at specified intervals. Samples of the tissue homogenate and the blood culture specimens were serially diluted in sterile PBS, plated on CLED agar and incubated for 24 hours at 37C. Antimicrobial treatment of infected mice. After infection the mice were treated with MAb V9-1 alone, cefsulodin alone, or both MAb V9-1 and cefsulodin. The control mice were not

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2096

HUMAN MAB AGAINST P. AERUGINOSA PYELONEPHRITIS

treated after infection. MAb V9-1 at a dose of 10.0 pg. was given 6 hours after infection and was repeated at the same dose every 24 hours for 3 days. A suboptimal dose of cefsulodin was chosen as 5.0 mg./kg./day on the basis of a preliminary experiment (0.6 X 50% protective doses) and was given 2 hours before the administration of MAb. Opsonophagocytic killing assays. Opsonophagocytic killing of P. aeruginosa by PMNs following incubation with or without 1/4 MIC of cefsulodin was determined in the presence or absence of MAb V9-1. The absorbed normal human serum (AbsNHS) was used as a complement source. Phagocytic mixtures containing PMNs, AbsNHS and bacterial suspension (with or without MAb V9-1) were prepared and incubated with constant shaking for 2 hours at 37C. Fluorescence-activated cell sorter analysis. Fluorescenceactivated cell sorter (FACS) was used for analysis of MAb V9-1 binding to bacteria. Bacteria were grown in trypticate soy broth with or without lJ4 MIC of cefsulodin for 18 hours at 37C with constant shaking. Those bacteria were then mixed with MAb V9-1 and incubated for 30 minutes. After incubation, bacteria were mixed with FITC-labeled goat antihuman IgM antibody. The final bacterial pellet was fixed with 1% paraformaldehyde and 10,000 bacteria were evaluated in a FACScan flow cytometer (Becton Dickinson, Mountain View, California). Statistical analysis. The significant differences in mouse survival and bacterial numbers in the blood and kidney were analyzed by the Fisher's exact probability test and the Kruskal-Wallis test. RESULTS

Therapeutic effects of MAb and cefsulodin on infected mice. In the first experiment using PA103 (serotype El,the dose of Wfsulodin was fixed at 5.0 mg.kg./day and the MAb V9-1 dose ranged from 0.1 to 10.0 fig. per mouse. After inoculation of bacteria into the bladder, the control mice receiving only cefsulodin showed a low survival rate that was approximately 20.0%. In the combination therapy groups, the survival rate of infected mice increased with an increase in MAb dose and reached 90.0%at 10.0 pg. of MAb V9-1 (fig. 1). The effects of combination therapy using 10.0 pg. of MAb v9-1 were further evaluated. The control mice that received no treatment had a survival rate of 16.1%after infection. The Survival rates for the mice receiving MAb V9-1 done or a suboptimal dose of cefsulodin alone were estimated to be

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FIG. 1. Effects of MAb Vg-1 in combination with Cefsulodin on BU-vival of mice infected with P. aeruginosa (10 mice per p u Mice were infected with p. aeruginosa PA103 and treated with 0 0,1 (0).1.0 (o), or 10.0 (B) pgJmoudday of MAb V9-1 In combmation with 5 mgJkgJday of cefsulodin for 3 days.

I 6

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35.7%and 25.08, respectively, and there was no significant difference between them. However, the survival rate for combination therapy (26 of 30 = 86.7%)was higher than those of mice receiving a single dose of MAb or cefsulodin (p <0.01) (fig. 2). All dead mice had bacteremia. In contrast, no increased survival rate was observed when the mice were infected with the P5 strain (serotype A). There was no significant difference between the combination therapy group and other groups (fig. 3). Antibacterial effect o f MAb and cefsulodin in the urinary tract infection of mice. At 24 hours after infection with PA103, the numbers of organisms in the kidneys of the control mice reached approximately 3.6 x lo6 CFU per kidney and bacteremia was also seen in their blood. When 10.0pg. of MAb or a suboptimal dose of cefsulodin was given separately, there was no reduction of the bacteria in the kidney and blood. However, the combination therapy with MAb V9-1 and cefsulodin resulted in a significant decrease in bacteria in the kidney (p <0.01) and blood (p <0.05).Although the P5infected mice were also treated with the combination therapy, the number of bacteria in the kidney reached approxi-

0 control 4

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treated with suboptimal dose (5 mgJkgJday) of cefsulodin alone 0, n = 28), MAb V9-1(10 H./mousdday) alone (0,n = 28). or both (B, n = 30) after intraurethral challenge with PA103. Treatments were continued for 3 days after infection.

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HUMAN MAB AGAINST P. AERUGINOSA PYELONEPHRITIS

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TABLE1. Viable bacterial cell numbers in the kidney and blood in mice challenged with P. aeruginosa PA103 and numbers of infected mice Treatment

P.aeruginosa (semtype)

MAb v9-1"

Viable bacterial cell numbers

Cefsulodinb

Kidnev (Lot? cfu/kidney)

Blood (Log cWml.)

1.2 2 1.3 1.02 1.2 0.2 2 0.6

+

5.6 I+_ 2.2 4.82 3.1 4.9 2 2.8 1.4 2 2.1' 4.82 2.1 4.6 ? 2.7 4.6 2 2.5 4.5 2 2.4

+

N D d

1.3 :1.1 1.2 Z 1.3 1.0 2 0.9 1.2 2 1.0

Numbers of infected mice belonephritis

12/13 9112 11/14 F~116~ 819 7l9 9/10 9111

Bacteremia

8/13 7/12 2/14 0/16e 6/9 519 6/10 7111

Values are mean 2 SD. ND = not detected. a MAb V9-1 at a dose of 10.0 mg. per mouse was administered intraperitonealy at 6. 30 and 54 hours after challenge. Suboptimal dose ( 5 mg.&g.) of cefsulodin was given at 4,28 and 52 hours afkr challenge. compared with other groups. Significantly different (p ~0.01) Significantly different (p (0.05) compared with untreated group. 'Significantly different (p <0.01)compared with untreated group.

mately 3.2 x lo4 CFU per kidney; the difference was not significant compared with the control (table 1). Opsonophagocytic killing assays. The opsonophagocytic killing activity was determined by the decrease in bacterial number and expressed as a percent of viable cells. Human PMNs showed increased opsonophagocytic killing activity against PA103 in the presence of MAb V9-1 compared with control at 2 hours after incubation. However, when bacteria were pretreated with L/4 MIC cefsulodin, this opsonophagocytic killing activity was significantly enhanced (p <0.05,two sample t test) and was observable a t 30 minutes after incubation (table 2). Fluorescence activated cell sorter analysis. Pseudomonas aeruginosa PA103 incubated with 1/4 MIC of cefsulodin showed increased MAb V9-1 binding compared with untreated bacteria (6g.4, A). Dual-parameter FACS analysis was also carried out to investigate the correlation between cekulodin-induced enlargement of bacterial size and MAb binding capacity. When the bacteria were not exposed to cefsulodin, the percentages of bacteria with small size and low MAb binding capacity and those with large sue and high MAb bindmg capacity were 77.31% and 9.25%, respectively. On the other hand, when the bacteria were exposed to the sub-MIC of cefsulodin the percentage changed to 41.85% and 24.17% (fig. 4, B and C). Thus pretreatment of bacteria with sub-MIC of cefsulodin increased bacterial size and binding capacity to MAb V9-1. DISCUSSION

This study demonstrated that the combination therapy of MAb V9-1 with cefsulodin was more effective in protecting the urinary tract against infection by P. aeruginosa than either therapy alone. It has been demonstrated that combination therapy with MAb and an antimicrobial agent is useful in treating P. aeruginosa pneumonia in neutropenic hosts.4 The urinary tract is considered as important a portal of bacterial entry into the body as the respiratory tract.7 Upper urinary tract infections may be caused by ascending infections after lower TABLE2. Effect of MA6 V9-I and cefsulodin combination therapy on oDsonoDhaaocvtic assays % Viable bacteria aRer incubation

Treatment

30 min.

60 min.

120 min.

Nontreated 64.2 2 14.1 34.9:14.8 12.62 3.3 Cefsulodin (U4MIC) 58.3 2 15.3 37.7 Z 23.7 9.8 2 6.1 40.0 t 13.9 15.8 2 5.7 MAb V9-1(50Mml.1 3.9 2 2.0' MAb V9-1and cefsulodin 14.7 2 6.8" 0.5 2 0.3d 6.32 l.lb Significantly different (p (0.05) compared with other groups. ) with other groups. Significantly different (p ~ 0 . 0 5compared Significantly different (p <0.05)compared with untreated group. Significantly different ( p <0.05)compared with other groups.

urinary tract manipulation and may result in pyelonephritis.8 Pseudomonas urinary tract infections, whose pathogenicity depends on host conditions, can develop into systemic infection that may result in lethal comp1ications.g In particular, the use of drugs that cause neutropenic conditions will predispose patients to severe renal infection and bacteremia.10.11 The neutropenic mouse is a model of infection in which bacteremia is achieved from colonized bacteria in the kidney by evasion of the host defense mechanisms. The present study, using this model, demonstrated that MAb V9-1 combined with cefsulodin inhibited the bacteria from growing in the kidney and invading the bloodstream. With regard to the mechanisms that cause the high therapeutic effect of this combined treatment, the local immune response to bacterial 0 antigen seems to be involved. The antibody to 0 antigen resulting from the immune response in the urinary tract is considered of some importance for protection.12-14 The MAbs specific for the 0 side chain (0 antigen) may act as one of the host defense mechanisms because it was shown to have opsonophagocytickilling activity of the corresponding serotype strains.3.4.15.16 Recently, the stable cell lines producing human MAbs specific for the several types of 0 antigens of P. aeruginosa were developed.6 For clinical use, the blend of several types of MAbs may be more useful than plasma-derived human immunoglobulins. However, the use of MAb alone was shown to be insufficient for effective treatment of experimental P. aeruginosa pneumonia in neutropenic hosts,4,6 which suggests that efXcient phagocytosis was not achieved by impaired neutrophils. We demonstrated that MAb V9-1 had much stronger protective activity in vitro and in vivo when combined with a suboptimal dose of cefsulodin. As for the mechanism by which antibiotics enhance the effect of MAb, several possibilities have been considered. Lorian and Atkinson17 indicated that P. aeruginosa exposed to sub-MIC of cefsulodin caused their elongation into filaments that show increased susceptibility to neutrophils. Therefore a combination of MAb and cefsulodin may be more efficacious in opsonophagocytickilling than either agent used individually. Another possible mechanism is that the antibiotics may alter the bacterial cell wall, such that 0 antigen epitope may be disclosed on the surface of the outer membrane. It is possible that the morphologic change of bacteria caused by antibiotics may permit easier access of the antibody to its target.18 Our FACS analysis of the size and antibody binding to P. aeruginosa may have proven the above possibility. Enhanced binding of MAb V9-1 to cefsulodin-modified bacteria was associated with alterations in bacterial cell morphology. Obviously P. aeruginosa exposed to sub-MIC of cefsulodin showed increased MAb binding to the bacteria accompanied

HUMAN MAB AGAINST P. AERUGINOSA PYELONEPHRITIS

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However, further studies are needed to clarify whether present combined chemotherapy is also applicable to Pseudomonas urinary tract infections in humans. Such studies are now in progress. REFERENCES

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FIG. 4. Fluorescence-activated cell sorter analysis of MAb V9-1 binding to untreated and cefsulodin-treated bacteria (A). After incubation for 2 hours with cefsulcdin (.....I. 18 hours with cefsulodin (......). or 18 hours without cefsulodin (-), MAb V9-1binding was detected with a n FITC-labeled goat anti-human antibody. Dualparameter FACS analysis of control bacteria ( B ) apd cefsuloenexposed bacteria (C) was also carried out. Here abscissa shows intensity of fluorescence and ordinate shows size of bacteria.

by enlargement in cell size (fig. 4, C). In contrast, there seems to be no correlation between cell size and the MAb binding capacity of bacteria that were not exposed to cefsulodin (fig. 4, B). We also find a significant effect in opsonophagocytic killing by human PMNs with MAb V9-1 for the cefsulodinexposed bacteria. Cefsulodin may permit the bacteria to be tied with more MAb so that PMNs can exert efficient phagocytosis.

1. Hector, R. F., Collins, M. S. and Pennington, J. E.: Treatment of experimental Pseudomonas aeruginosa pneumonia with a human IgM monoclonal antibody. J. Infect. Dis., 160: 483, 1989. 2. Oishi, K,Sonoda, F., Miwa, H., Tanaka, H., Watanabe, K, Matsumoto, K and Pollack, M.: Pharmacodynamic and protective properties of a murine lipopolysaccharide-specific monoclonal antibody in experimental Pseudomonas aeruginosa in mice. Microbiol. Immunol., 35: 1131,1991. 3. Pennington, J. E.,Small, G. J., Lostrom, M. E. and Pier, G. B.: Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia. Infect. Immun., 54.239, 1986. 4. Oishi, K,Sonoda, F.. Iwagaki, A., Kobayashi, S., Nagatake, T. and Matsumoto, K: Effects of the combination of lipopolysaccharide-specificmonoclonal antibodies and sparfloxacin against Pseudomonas aeruginosa pneumonia in neutropenic mice. Antimicrob. Agents Chemother., 38:1352,1992. 5. Pennington, J. E. and Small, G. J.: Passive immune therapy for experimental Pseudomonas aeruginosa pneumonia in the neutropenic host. J. Infect. Dis., 166 973, 1987. 6. O'Oka, H., Chonan, E., Mizutani, K, Fukuda, T., Kuroiwa, Y., Ono, Y. and Shigeta, S.: Establishment of stable cell Lines producing anti-Pseudomonas aeruginosa monoclonal antibodies and their protective effects for the infection in mice. Microbiol. Immunol., 36: 1305,1992. 7. Hilf, M., Yu,V. L., Sharp, J., Zuravleff, J. J., Korvick, J. A. and Muder, R. R.: Antibiotic therapy for Pseudomonas aeruginosa bacteremia: outcome correlations in a prospective study of 200 patients. Am. J. Med., 81:540, 1989. 8. Moore, B. and Forman, A.: An outbreak of urinary Pseudomonas aeruginosa infection acquired during urological operation. Lancet, II:929, 1966. 9. Epstein, M.: Aerobacter aerogenes bacteremia: control of a lethal complication. J. Urol., 91: 600,1965. 10. Lyon, D., Howard, E. B. and Montgomerie, J. Z.: Increased severity of urinary tract infection and bacteremia in mice with urinary bladder injury induced by cyclophosphamide. Infect. Immun., 38:558,1982. 11. Montgomerie, J. Z., Tuddenham, W. J., Howard, E. B. and Morrow, J. W.: Pseudomonas urinary tract infection in mice. Infect. Immun., 2% 267, 1980. 12. Kaijser, B., Larsson, P. and Olling, S.: Protection against ascending Escherichia coli pyelonephritis in rats and significance of local immunity. Infect. Immun., 2 0 78, 1978. 13. Lehmann, J. A., Smith, J. W., Miller, T. E., Barnett, J. A. and Sanford, J. P.: Local immune response in experimental pyelonephritis. J. Clin. Invest., 47: 2541,1968. 14. Smith, J. W. and W s e r , B.: The local immune response to Escherichia coli 0 and K antigen in experimental pyelonephritis. J. Clin. Invest., 58: 276,1976. 15. Sawada, S.,Kawamura, T. and Masuho, Y.: Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa. J. Gen. Microbiol., 133: 3581,1987. 16. Zweerink, H.J.,Detolla, L. J., Gammon, M. C., Hutchison, C. F., Puckett, J. M. and Sigal, N. H.: A human monoclonal antibody that protects mice against Pseudomonas-induced pneumonia. J. Infect. Dis., 162 254, 1990. 17. Lorian, V. and Atkinson, B.: Bactericidal effect of polymorphonuclear neutrophils on antibiotic-induced filaments of gramnegative bacilli. J. Infect. Dis., 149 719, 1984. 18. Collins, H. H., Cross,A. S., Dobek, A., Opal, S. M., McClain, J. B. and Sadoff, J. C.: Oral ciprofloxacin and a monoclonal antibody to lipopolysaccharide protect leukopenic rats from lethal infection with Pseudomonas aeruginosa. J. Infect. Dis., 159 1073, 1989.