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Treatment of feline leukemia virus-infected cats with paramunity inducer
Veterinary Immunology and Immunopathology 65 (1998) 267±275
Treatment of feline leukemia virus-infected cats with paramunity inducer K. Hartmanna,*, A. Blocka, G. Ferka, A. Vollmarb, M. Goldbergc, H. Lutzd a
I. Medizinische Tierklinik, Ludwig-Maximilians-UniversitaÈt, Munich, Germany Institut fuÈr Pharmakologie, Ludwig-Maximilians-UniversitaÈt, Munich, Germany c Institut fuÈr Physiologie, Ludwig-Maximilians-UniversitaÈt, Munich, Germany d Department of Medicine, School of Veterinary Medicine, University of Zurich, Zurich, Switzerland b
Abstract Two placebo-controlled double-blind trials were performed to determine the therapeutic efficacy of the paramunity inducer, Baypamun1 in feline leukemia virus (FeLV) -infected cats under controlled conditions. In the first study, 120 cats were involved; 60 cats were treated with Baypamun1 and 60 with a placebo preparation of virus-free cell culture medium. Dosage and administration of the drug over a 7-week period were performed according to the instructions given by the company. Remission of viremia occurred in 12% and 7% of the cats treated with Baypamun1 and placebo, respectively. This difference was not statistically significant. In the second study, 30 naturally infected cats were treated in a placebo-controlled double-blind trial. In total, 20 immunological, clinical, laboratory, and virological parameters were examined. No statistically significant differences could be demonstrated between Baypamun1 and placebo application. Therefore, FeLV infection was not influenced by Baypamun1 treatment. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Feline Leukemia virus; Paramunity inducer
1. Introduction A sensation was caused when HoÈrber and Mayr (HoÈrber and Mayr, 1991; HoÈrber et al., 1992; Mayr and HoÈrber, 1992) reported that they were able to cure 80±100% FeLV* Corresponding author. Abbreviations: BRM, biologic response modifier; ELISA, enzyme-linked immunosorbent assay; FeLV, feline leukemia virus; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; p, statistical significance; PCV, packed cell volume; RIA, radio-immunoassay; R-PE, R-phycoerythrin; TCID, tissue culture infectious dose; UV, ultraviolet 0165-2427/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 8 ) 0 0 1 6 1 - 5
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infected cats from viremia by using an immunomodulating compound. Articles in cat breeder and cat owner journals appeared indicating that there is a cure for FeLV-infected cats suffering from this deadly infection. The immunomodulator used in those studies was the so-called `paramunity inducer' PIND-ORF (Baypamun1, Bayer, Leverkusen, Germany) which consists of inactivated parapox ovis virus. Baypamun1 is the most commonly used drug for treatment of FeLV infection in Germany and other European countries. Therapy and prophylaxis with biological response modifiers (BRM), immunomodulators, or paramunity inducers have recently become more attractive in veterinary medicine. Effects of BRM on unspecific defense functions are difficult to test and the results (positive and negative) in in vitro test systems cannot simply be transferred to in vivo situations and vice versa (BuÈttner and Mayr, 1986). Since Edward Jenner's initiation of the century of vaccination, immunomodulation has a historical background (Baxby, 1981). During the worldwide smallpox eradication campaign experience with vaccinia virus vaccines was gained. The observations included harmful side-effects as well as beneficial effects. Therefore, it is advisable to choose the term `immunomodulation' for vaccinia virus vaccination side-effects because they can be immunosuppressive or immunostimulative. The harmful side-effects indicate that most of the microorganisms harbor the potency for initial immunosuppression. Meanwhile, immunosuppressive effects and evasion of immune response have been determined on a molecular basis for vaccinia virus and other poxviruses (Gooding, 1992). In contrast to postvaccinational complications, beneficial side-effects have also been observed. They have been reported mainly associated with sudden resolution of certain diseases after vaccinia virus vaccination. Thus, improved healing of herpes virus-induced signs, papillomatosis, chronic dermal diseases, infections of the upper respiratory tract, or even tumors have been reported. Mayr (1982) tried to find a general term for non-specific immune reactions and called the phenomenon `paramunity.' This name expresses that something happens besides (`para') and before immunization (BuÈttner, 1993). Mayr and BuÈttner (1984) defined `paramunity as a non-specific protective mechanism against various infections, toxins, detrimental substances or xenogenic as well as transformed syngenic cells. Paramunization includes all measures to induce iatrogenic paramunity. Inducers of paramunity are drugs whose function is to elicit protective and defense mechanisms in man and animal' (Mayr and BuÈttner, 1984). The aim of this investigation was to determine if Baypamun1 was helpful for effective therapy of FeLV-infected cats in two placebo-controlled double-blind studies using statistical evaluation for objective interpretation of a therapeutic effect. 2. Materials and methods 2.1. Compounds The paramunity inducer Baypamun1 (PIND-ORF, Bayer, Leverkusen, Germany) was administered at a dosage of 1 ml per cat, independent of the body weight. One dose
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resuspended in 1 ml contained at least 106.45 TCID50 lyophilized parapox ovis strain D 1701 (chemically inactivated) and polygelatine as stabilizer (25.0 mg). The host system was a bovine kidney cell line. The placebo contained resuspended lyophilized supernatant of uninfected bovine kidney cell cultures and was also administered in a dosage of 1 ml per cat. Both compounds were coded and could not be distinguished by any investigator or participant of the studies. 2.2. Cats In the first study, 120 naturally FeLV-infected cats were included. The cats were randomly divided in two equal groups, 60 cats receiving Baypamun1, and 60 receiving placebo. The second study contained 30 naturally FeLV-infected cats with one-half being randomly classed in the Baypamun1 group, and the other half in the placebo group. 2.3. Diagnosis of FeLV infection FeLV infection was diagnosed using the enzyme-linked immunosorbent assay (ELISA) PetChek FeLV1 (IDEXX, Maine) which detects FeLV p27 antigen in serum samples. To avoid inclusion of false-positive individuals, cats were only considered FeLV-positive if the serum was confirmed positive in three independent ELISA of at least two different samples. 2.4. Design of the studies Both studies were designed as placebo-controlled double-blind studies. The period of the treatment was standardized to 7 weeks. In the first week cats received the compounds twice every other day, later on once a week. The compounds were injected subcutaneously a dosage of 1 ml per cat. 2.5. Parameters to control treatment efficacy In study I, 120 cats were treated. Parameters monitored and evaluated in study I included the general clinical status, weight change, number of cats converting to FeLVnegative, and concentration of FeLV p27 antigen. Study II included 30 cats. Parameters evaluated in study II were improvement of respiratory tract disorders, rhinitis, stomatitis, weight change, index of Karnofsky, hemoglobin value, packed cell volume (PCV), relative and absolute CD4 counts, relative and absolute CD8 counts, CD4/CD8 ratio, neopterin concentration in serum measured by radio-immunoassay (RIA), 7-Xanthopterin concentration in serum and urine measured by high pressure liquid chromatography (HPLC), biopterin concentration in serum and urine measured by HPLC, number of cats converting to a FeLV-negative state, concentration of FeLV p27 antigen, and survival rate.
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Table 1 Scores of symptoms for the status of the oral cavity Degree of stomatitis 0 1 2 3 4 5 6 7 8 9 10
No clinical signs Minor redness of the gingiva around the teeth Mean degree of redness of the mucous membrane Deep redness, minor swelling of the mucous membrane Deep redness, extended swelling of the mucous membrane Deep redness, strong swelling of the mucous membrane, some proliferation Deep redness, strong swelling of the mucous membrane, prominent proliferation Deep redness, strong swelling of the mucous membrane, proliferation and some erosions Deep redness, strong swelling of the mucous membrane, proliferation, erosions, occasional bleeding caused by manipulations Deep redness, strong swelling of the mucous membrane, proliferation, erosions, spontaneous bleeding Deep redness, strong swelling of the mucous membrane, proliferation, deep ulceration, bleeding
2.6. Clinical parameters Clinical markers were judged once a week. Every symptom was classified in 10 `scores of symptoms' as shown for the status of the oral cavity (Table 1) which is a frequent clinical sign in FeLV-infected cats. The degree of improvement regarding these signs was defined as the score of symptoms at the end of the treatment minus the score of symptoms at the beginning. Once a week, the weight of all cats was determined. Changes of weight during the treatment period were registered. To evaluate the quality of life of the cats the index of Karnofsky developed for human cancer patients (Karnofsky et al., 1948) was modified for cats (Hartmann et al., 1997). Once a week, the Karnofsky's score was monitored in the cats. 2.7. Laboratory parameters Leukocyte counts and hemoglobin values were evaluated weekly in an automatic blood cell counter (Sysmex Microcounter E-3000, Digitana, Hamburg, Germany). The PCV was determined in microhematocapillary tubes after a 7 min centrifugation at 1000 rpm (MikrohaÈmatokritzentrifuge, Hettich, Tuttlingen, Germany). To differentiate the blood cells, blood spreads were stained (Merz and Dade, DuÈdingen, Switzerland) and manually counted. Liver and pancreatic enzymes, electrolytes and kidney parameters were checked to monitor side-effects of the treatment. They were determined as described previously (Hartmann, 1989). 2.8. Lymphocyte subpopulations Absolute and relative CD4 and CD8 counts and the CD4/CD8 ratio were determined at the beginning, in the middle, and at the end of the treatment period. The lymphocyte
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subpopulations were stained by reaction with a fluorescein isothiocyanate (FITC)conjugated monoclonal mouse anti-feline pan T-cell (anti-CD3) antibody, a FITCconjugated monoclonal mouse anti-feline CD4 antibody, and a R-phycoerythrin (R-PE) -conjugated monoclonal mouse anti-feline CD8 antibody (Southern Biotechnology Associates, Birmingham). The fluorescence-tagged cell populations of CD4 and CD8 cells were quantitatively determined by flow cytometry with a FACScan Cytometer using Cell Quest 1.1.1. software (Becton Dickinson, Heidelberg, Germany). Absolute lymphocyte counts were calculated of the total leukocyte cell counts and differential blood cell counts. 2.9. Neopterin serum concentration Neopterin acts as surrogate marker for progression of disease and treatment efficacy in anti-human immunodeficiency virus therapy (Fuchs et al., 1988; Reddy et al., 1991). The RIA IMMUtest1 Neopterin (BRAHMS Diagnostica, Berlin, Germany) containing sheep anti-neopterin antibodies and radioactive-labeled tracer was used to determine the neopterin concentration in the cat serum. 2.10. Concentration of other pterins in serum and urine Since only humans and primates show high neopterin serum values (Goldberg et al., 1989), other pterins were monitored additionally. Biopterin and 7-Xanthopterin were shown to be most valuable among the pterins in cats (Hartmann, 1995) and therefore were chosen for evaluation in this study following a procedure as described previously (Goldberg et al., 1989). All urine and blood samples were prepared under light protection. Identification of each pterin was based on its characteristic ultraviolet (UV) absorption and fluorescence spectrum and quantified by HPLC and electrochemical detection. The stationary low polar phase contained a 250 or 500 mm 5 mC 18 Silika ODS2-column with a 4.6 inner diameter (Spherisorb±Octodecylsilan 2, Grom, Herrenberg±Kayh, Germany). The mobile phase was a high polar mixture of 10% methanol, 1% acetonitrile, 1.5 mmol lÿ1 0.01% phosphonic acid in milli-Q water. Determination of the pterins took place using an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Pterin concentrations in the samples were determined in comparison to standard pterins (Schricks, Wettswil, Switzerland). 2.11. Concentration of FeLV p27 antigen The ELISA for quantitative determination of FeLV p27 antigen was developed and described by Lutz et al. (1983). 2.12. Statistical evaluation Statistically significant differences between the Baypamun1 and the placebo groups in all of the parameters monitored were evaluated by Student's t-test (laboratory, immunological, and virological parameters), Mann±Whitney's U-test (clinical para-
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meters), and Chi-square test (conversion to FeLV-negative and survival rate). Statistical significance (p) was fixed at 0.05. 3. Results 3.1. Study I In study I, 120 naturally FeLV-infected cats were treated; 60 cats received Baypamun1, 60 received placebo. Seven of the 60 cats (11.7%) treated with Baypamun and four of the 60 cats (6.7%) receiving placebo converted to a FeLV-negative state during the 6-week therapy interval (Table 2). The difference in the number of converting cats was not statistically significant (p0.343). The mean course of the serum FeLV p27 antigen concentrations during the treatment in study I is shown in Fig. 1. On average the antigen concentration decreased by 7.2% in the cats of the Baypamun1 group and by 5.5% in the placebo group. No significant difference could be shown (p0.851). The general clinical status improved in 47% and 33% of the cats receiving Baypamun1 and placebo, respectively. In the Baypamun1 group, 32% of the cats gained weight, while in the placebo group only 27% increased in body weight. Neither Table 2 Percentage of cats converting to FeLV-negative and mean change of the concentration of FeLV p27 antigen during study I in the cats receiving Baypamun1 and in the cats receiving placebo, and p-value of the differences between the two groups
Conversion to FeLV-negative (%) Concentration of FeLV p27 (%)
Baypamun1
Placebo
p
11.7 ÿ7.2
6.7 ÿ5.5
0.343 0.851
Fig. 1. Mean changes of the serum FeLV p27 antigen concentration during the treatment in study I.
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Fig. 2. Mean changes of the serum FeLV p27 antigen concentration during the treatment in study II.
the differences in the general clinical status (p0.253) nor in the weight change (p0.584) were statistically significant. 3.2. Study II In study II, 30 FeLV-infected cats were treated, 15 cats received Baypamun1, and 15 received placebo. Remission of viremia occurred in two cats of each group (15.4%). The FeLV antigen level was decreased by 1.9% on average after Baypamun1 treatment and increased by 3.7% on average in the placebo recipients (Fig. 2). The difference was not statistically significant (p0.842). Twenty parameters including improvement of lower respiratory tract disorders, rhinitis, stomatitis, weight change, index of Karnofsky, hemoglobin value, PCV, relative and absolute CD4 counts, relative and absolute CD8 counts, CD4/CD8 ratio, neopterin concentration in serum measured by RIA, 7-xanthopterin concentration in serum and urine measured by HPLC, biopterin concentration in serum and urine measured by HPLC, number of cats converting to FeLV-negative, concentration of FeLV p27 antigen, and survival rate were evaluated (Table 3). In none of these parameters could a statistically significant difference could be demonstrated between Baypamun1 and placebo application. 4. Discussion There was no statistically significant difference regarding any of the investigated immunological, clinical, laboratory, or virological parameters between animals receiving Baypamun1 or placebo neither in the first nor second study. In 11 cats of study I and four cats of study II, there was a conversion from a FeLV-positive to a FeLV-negative state but the difference in number of cats was not statistically significant between the groups. These cats were generally younger than 1 year and had no clinical signs. It can be assumed that they were in a state of transient viremia.
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Table 3 Mean changes of the evaluated parameters during study II in the cats receiving Baypamun1 and in the cats receiving placebo, and p-value of the differences between the two groups Baypamun1 Lower respiratory disorders (score) Rhinitis (score) Stomatitis (score) Weight change (kg) Index of Karnofsky (score) Hemoglobin value (g dlÿ1) PCV (%) Relative CD4 count (%) Absolute CD4 count (cells mlÿ1) Relative CD8 count (%) Absolute CD8 count (cells mlÿ1) CD4/CD8 ratio Neopterin in serum (nmol lÿ1) 7-Xanthopterin in serum (nmol lÿ1) 7-Xanthopterin in urine (nmol mmolÿ1)a Biopterin in serum (nmol lÿ1) Biopterin in urine (nmol mmolÿ1)a Conversion to FeLV-negative (%) Concentration of FeLV p27 (%) Survival rate (%)
ÿ0.5 ÿ0.4 ÿ1.5 0.1 8.1 ÿ1.3 ÿ0.1 ÿ3.3 82.2 0.0 39.4 ÿ0.4 ÿ6.6 0.9 0.0 ÿ20.0 ÿ0.1 15.4 ÿ1.9 33.3
Placebo
p
0.1 ÿ0.2 ÿ0.3 0.2 ÿ1.3 1.1 0.1 ÿ1.1 6.1 ÿ0.5 24.6 0.3 ÿ2.5 ÿ0.9 ÿ0.7 1.8 0.0 15.4 3.7 60.0
0.338 0.779 0.481 0.140 0.145 0.851 0.797 0.964 0.810 0.343 0.590 0.744 0.326 0.375 0.100 0.129 0.163 1.000 0.842 0.143
a
7-Xanthopterin and biopterin in urine were measured in nmol mmolÿ1 urine creatinine.
As shown in these studies, there was no cure of FeLV infection by the paramunity inducer Baypamun1. There is no explanation of the divergent results by HoÈrber and Mayr (HoÈrber and Mayr, 1991; HoÈrber et al., 1992; Mayr and HoÈrber, 1992) who stated that they were able to cure 80±100% FeLV-infected cats from viremia by using Baypamun1. Certainly, an accurate diagnosis of FeLV infection is a prerequisite for reliable results. For this reason we considered cats FeLV-positive when the serum was tested positive in three independent ELISA of at least two different samples to avoid inclusion of false-positive animals. Especially in study II, we tried to take a lot of parameters into consideration. It was shown that most of the clinical parameters, the general clinical status, and the quality of life, measured by the index of Karnofsky, improved on average in both groups. This was due to two reasons. Firstly, the cats were presented to the hospital for injections and investigations very often and therefore were under excellent veterinary care during the treatment period. Secondly, supportive management and treatment of secondary infections were provided to all cats. For proper interpretation of the results, comparison with a placebo group is unavoidable. To prevent the subjective influence of the investigator, the study was also double-blind so that objective improvement could be determined. In these two studies, no side-effects of Baypamun1 treatment were observed. However, there was also no detectable influence of Baypamun1 treatment on the FeLV infection. Thus, Baypamun1 treatment cannot be recommended for treatment of FeLV
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infection. The importance of conducting treatment studies as placebo-controlled doubleblind trials was demonstrated in this investigation. Acknowledgements The authors thank Dr. Christian Leutenegger and Dr. Erich Lederer for their advice and help. This research was supported by the Human Capital and Mobility Programme of the European Commission and by Bayer, Leverkusen, Germany. References Baxby, D., 1981. Jenner's Smallpox Vaccine. Morrison and Gibb, London. BuÈttner, M., Mayr, A., 1986. Tests on protection against viral diseases. Comp. Immun. Microbiol. Infect. Dis. 9, 205±215. BuÈttner, M., 1993. Principles of paramunization, option and limits in veterinary medicine. Comp. Immun. Microbiol. Infect. Dis. 18, 1±10. Fuchs, D., Unterweger, B., Hausen, A., Reibnegger, G., Werner, E.R., Hengster, P., Hinterhuber, H., Dierich, M.D., Wachter, H., Blattner, W.A., Weiss, S.H., 1988. Anti-HIV-1 antibodies, anti-HTLV-1 antibodies and neopterin levels in parenteral drug addicts in the Austrian Tyrol. J. Acquir. Immune Defic. Syndr. 1, 65±66. Goldberg, M., Gassner, F., Singer, L.J., Merkenschlager, M., 1989. Pteridinkonzentrationen in Harn und Organgeweben von Hund und Katze bei verschiedenen Neoplasien und Virusinfektionen. TieraÈrztl. Prax. 5, 37±41. Gooding, L.R., 1992. Virus proteins that counteract host immune defenses. Cell 71, 5±7. Hartmann, K., 1989. Referenzbereiche in der Labordiagnostik der Katze. TieraÈrztl. Prax. 5, 58±62. Hartmann, K., 1995. Entwicklung eines Testsystems zur Erprobung neuer Medikamente gegen die FIV-Infektion der Katze als Modell fuÈr die Behandlung erworbener ImmunschwaÈchesyndrome. Habil. med. vet., MuÈnchen. Hartmann, K., Kuffer, M., Kraft, W., 1997. Modifikation des Karnofsky-Indexes fuÈr die Katze zur Beurteilung der LebensqualitaÈt and Therapiekontrolle. TieraÈrztl. Prax. 25, 540±542. HoÈrber, D., Mayr, B., 1991. Paramunisierung FeLV-positiver Katzen mit PIND-AVI. TieraÈrztl. Prax. 19, 311± 314. HoÈrber, D., Schnabl, W., Mayr, B., 1992. Praxiserfahrungen bei der Paramunisierung FeLV-positiver Katzen mit Baypamun HK. TieraÈrztl. Umschau 47, 556±560. Karnofsky, D.A., Abelmann, W.H., Craver, L.F., Burchenal, J.H., 1948. The use of the nitrogen mustard in the palliative treatment of carcinoma. Cancer, 634±656. Lutz, H., Pedersen, N.C., Durbin, R., Theilen, G.H., 1983. Monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27. J. Immuno. Met. 56, 209±220. Mayr, A., BuÈttner, M., 1984. Neue Erkenntnisse uÈber die Grundlagen der ParamunitaÈt und Paramunisierung. Berl. MuÈnch. TieraÈrztl. Wschr. 97, 429±435. Mayr, A., 1982. ParamunitaÈt und Paramunisierung. J. Vet. Med. B 29, 5±23. Mayr, B., HoÈrber, D., 1992. Paramunisierung FeLV-positiver Katzen ± ein Bericht aus der Praxis. Kleintierprax. 37, 515±518. Reddy, M.M., McKinley, G.F., Grieco, M.H., 1991. Evaluation of HIV p24 antigen, beta2-microglobulin, neopterin, soluble CD4, soluble CD8, and soluble interleukin-2 receptor levels in patients with AIDS or AIDS-related complex treated with 20 ,30 -dideoxyinosine (ddI). J. Clin. Lab. Anal. 5, 396±398.
Treatment of feline leukemia virus-infected cats with paramunity inducer K. Hartmanna,*, A. Blocka, G. Ferka, A. Vollmarb, M. Goldbergc, H. Lutzd a
I. Medizinische Tierklinik, Ludwig-Maximilians-UniversitaÈt, Munich, Germany Institut fuÈr Pharmakologie, Ludwig-Maximilians-UniversitaÈt, Munich, Germany c Institut fuÈr Physiologie, Ludwig-Maximilians-UniversitaÈt, Munich, Germany d Department of Medicine, School of Veterinary Medicine, University of Zurich, Zurich, Switzerland b
Abstract Two placebo-controlled double-blind trials were performed to determine the therapeutic efficacy of the paramunity inducer, Baypamun1 in feline leukemia virus (FeLV) -infected cats under controlled conditions. In the first study, 120 cats were involved; 60 cats were treated with Baypamun1 and 60 with a placebo preparation of virus-free cell culture medium. Dosage and administration of the drug over a 7-week period were performed according to the instructions given by the company. Remission of viremia occurred in 12% and 7% of the cats treated with Baypamun1 and placebo, respectively. This difference was not statistically significant. In the second study, 30 naturally infected cats were treated in a placebo-controlled double-blind trial. In total, 20 immunological, clinical, laboratory, and virological parameters were examined. No statistically significant differences could be demonstrated between Baypamun1 and placebo application. Therefore, FeLV infection was not influenced by Baypamun1 treatment. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Feline Leukemia virus; Paramunity inducer
1. Introduction A sensation was caused when HoÈrber and Mayr (HoÈrber and Mayr, 1991; HoÈrber et al., 1992; Mayr and HoÈrber, 1992) reported that they were able to cure 80±100% FeLV* Corresponding author. Abbreviations: BRM, biologic response modifier; ELISA, enzyme-linked immunosorbent assay; FeLV, feline leukemia virus; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; p, statistical significance; PCV, packed cell volume; RIA, radio-immunoassay; R-PE, R-phycoerythrin; TCID, tissue culture infectious dose; UV, ultraviolet 0165-2427/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 8 ) 0 0 1 6 1 - 5
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infected cats from viremia by using an immunomodulating compound. Articles in cat breeder and cat owner journals appeared indicating that there is a cure for FeLV-infected cats suffering from this deadly infection. The immunomodulator used in those studies was the so-called `paramunity inducer' PIND-ORF (Baypamun1, Bayer, Leverkusen, Germany) which consists of inactivated parapox ovis virus. Baypamun1 is the most commonly used drug for treatment of FeLV infection in Germany and other European countries. Therapy and prophylaxis with biological response modifiers (BRM), immunomodulators, or paramunity inducers have recently become more attractive in veterinary medicine. Effects of BRM on unspecific defense functions are difficult to test and the results (positive and negative) in in vitro test systems cannot simply be transferred to in vivo situations and vice versa (BuÈttner and Mayr, 1986). Since Edward Jenner's initiation of the century of vaccination, immunomodulation has a historical background (Baxby, 1981). During the worldwide smallpox eradication campaign experience with vaccinia virus vaccines was gained. The observations included harmful side-effects as well as beneficial effects. Therefore, it is advisable to choose the term `immunomodulation' for vaccinia virus vaccination side-effects because they can be immunosuppressive or immunostimulative. The harmful side-effects indicate that most of the microorganisms harbor the potency for initial immunosuppression. Meanwhile, immunosuppressive effects and evasion of immune response have been determined on a molecular basis for vaccinia virus and other poxviruses (Gooding, 1992). In contrast to postvaccinational complications, beneficial side-effects have also been observed. They have been reported mainly associated with sudden resolution of certain diseases after vaccinia virus vaccination. Thus, improved healing of herpes virus-induced signs, papillomatosis, chronic dermal diseases, infections of the upper respiratory tract, or even tumors have been reported. Mayr (1982) tried to find a general term for non-specific immune reactions and called the phenomenon `paramunity.' This name expresses that something happens besides (`para') and before immunization (BuÈttner, 1993). Mayr and BuÈttner (1984) defined `paramunity as a non-specific protective mechanism against various infections, toxins, detrimental substances or xenogenic as well as transformed syngenic cells. Paramunization includes all measures to induce iatrogenic paramunity. Inducers of paramunity are drugs whose function is to elicit protective and defense mechanisms in man and animal' (Mayr and BuÈttner, 1984). The aim of this investigation was to determine if Baypamun1 was helpful for effective therapy of FeLV-infected cats in two placebo-controlled double-blind studies using statistical evaluation for objective interpretation of a therapeutic effect. 2. Materials and methods 2.1. Compounds The paramunity inducer Baypamun1 (PIND-ORF, Bayer, Leverkusen, Germany) was administered at a dosage of 1 ml per cat, independent of the body weight. One dose
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resuspended in 1 ml contained at least 106.45 TCID50 lyophilized parapox ovis strain D 1701 (chemically inactivated) and polygelatine as stabilizer (25.0 mg). The host system was a bovine kidney cell line. The placebo contained resuspended lyophilized supernatant of uninfected bovine kidney cell cultures and was also administered in a dosage of 1 ml per cat. Both compounds were coded and could not be distinguished by any investigator or participant of the studies. 2.2. Cats In the first study, 120 naturally FeLV-infected cats were included. The cats were randomly divided in two equal groups, 60 cats receiving Baypamun1, and 60 receiving placebo. The second study contained 30 naturally FeLV-infected cats with one-half being randomly classed in the Baypamun1 group, and the other half in the placebo group. 2.3. Diagnosis of FeLV infection FeLV infection was diagnosed using the enzyme-linked immunosorbent assay (ELISA) PetChek FeLV1 (IDEXX, Maine) which detects FeLV p27 antigen in serum samples. To avoid inclusion of false-positive individuals, cats were only considered FeLV-positive if the serum was confirmed positive in three independent ELISA of at least two different samples. 2.4. Design of the studies Both studies were designed as placebo-controlled double-blind studies. The period of the treatment was standardized to 7 weeks. In the first week cats received the compounds twice every other day, later on once a week. The compounds were injected subcutaneously a dosage of 1 ml per cat. 2.5. Parameters to control treatment efficacy In study I, 120 cats were treated. Parameters monitored and evaluated in study I included the general clinical status, weight change, number of cats converting to FeLVnegative, and concentration of FeLV p27 antigen. Study II included 30 cats. Parameters evaluated in study II were improvement of respiratory tract disorders, rhinitis, stomatitis, weight change, index of Karnofsky, hemoglobin value, packed cell volume (PCV), relative and absolute CD4 counts, relative and absolute CD8 counts, CD4/CD8 ratio, neopterin concentration in serum measured by radio-immunoassay (RIA), 7-Xanthopterin concentration in serum and urine measured by high pressure liquid chromatography (HPLC), biopterin concentration in serum and urine measured by HPLC, number of cats converting to a FeLV-negative state, concentration of FeLV p27 antigen, and survival rate.
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Table 1 Scores of symptoms for the status of the oral cavity Degree of stomatitis 0 1 2 3 4 5 6 7 8 9 10
No clinical signs Minor redness of the gingiva around the teeth Mean degree of redness of the mucous membrane Deep redness, minor swelling of the mucous membrane Deep redness, extended swelling of the mucous membrane Deep redness, strong swelling of the mucous membrane, some proliferation Deep redness, strong swelling of the mucous membrane, prominent proliferation Deep redness, strong swelling of the mucous membrane, proliferation and some erosions Deep redness, strong swelling of the mucous membrane, proliferation, erosions, occasional bleeding caused by manipulations Deep redness, strong swelling of the mucous membrane, proliferation, erosions, spontaneous bleeding Deep redness, strong swelling of the mucous membrane, proliferation, deep ulceration, bleeding
2.6. Clinical parameters Clinical markers were judged once a week. Every symptom was classified in 10 `scores of symptoms' as shown for the status of the oral cavity (Table 1) which is a frequent clinical sign in FeLV-infected cats. The degree of improvement regarding these signs was defined as the score of symptoms at the end of the treatment minus the score of symptoms at the beginning. Once a week, the weight of all cats was determined. Changes of weight during the treatment period were registered. To evaluate the quality of life of the cats the index of Karnofsky developed for human cancer patients (Karnofsky et al., 1948) was modified for cats (Hartmann et al., 1997). Once a week, the Karnofsky's score was monitored in the cats. 2.7. Laboratory parameters Leukocyte counts and hemoglobin values were evaluated weekly in an automatic blood cell counter (Sysmex Microcounter E-3000, Digitana, Hamburg, Germany). The PCV was determined in microhematocapillary tubes after a 7 min centrifugation at 1000 rpm (MikrohaÈmatokritzentrifuge, Hettich, Tuttlingen, Germany). To differentiate the blood cells, blood spreads were stained (Merz and Dade, DuÈdingen, Switzerland) and manually counted. Liver and pancreatic enzymes, electrolytes and kidney parameters were checked to monitor side-effects of the treatment. They were determined as described previously (Hartmann, 1989). 2.8. Lymphocyte subpopulations Absolute and relative CD4 and CD8 counts and the CD4/CD8 ratio were determined at the beginning, in the middle, and at the end of the treatment period. The lymphocyte
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subpopulations were stained by reaction with a fluorescein isothiocyanate (FITC)conjugated monoclonal mouse anti-feline pan T-cell (anti-CD3) antibody, a FITCconjugated monoclonal mouse anti-feline CD4 antibody, and a R-phycoerythrin (R-PE) -conjugated monoclonal mouse anti-feline CD8 antibody (Southern Biotechnology Associates, Birmingham). The fluorescence-tagged cell populations of CD4 and CD8 cells were quantitatively determined by flow cytometry with a FACScan Cytometer using Cell Quest 1.1.1. software (Becton Dickinson, Heidelberg, Germany). Absolute lymphocyte counts were calculated of the total leukocyte cell counts and differential blood cell counts. 2.9. Neopterin serum concentration Neopterin acts as surrogate marker for progression of disease and treatment efficacy in anti-human immunodeficiency virus therapy (Fuchs et al., 1988; Reddy et al., 1991). The RIA IMMUtest1 Neopterin (BRAHMS Diagnostica, Berlin, Germany) containing sheep anti-neopterin antibodies and radioactive-labeled tracer was used to determine the neopterin concentration in the cat serum. 2.10. Concentration of other pterins in serum and urine Since only humans and primates show high neopterin serum values (Goldberg et al., 1989), other pterins were monitored additionally. Biopterin and 7-Xanthopterin were shown to be most valuable among the pterins in cats (Hartmann, 1995) and therefore were chosen for evaluation in this study following a procedure as described previously (Goldberg et al., 1989). All urine and blood samples were prepared under light protection. Identification of each pterin was based on its characteristic ultraviolet (UV) absorption and fluorescence spectrum and quantified by HPLC and electrochemical detection. The stationary low polar phase contained a 250 or 500 mm 5 mC 18 Silika ODS2-column with a 4.6 inner diameter (Spherisorb±Octodecylsilan 2, Grom, Herrenberg±Kayh, Germany). The mobile phase was a high polar mixture of 10% methanol, 1% acetonitrile, 1.5 mmol lÿ1 0.01% phosphonic acid in milli-Q water. Determination of the pterins took place using an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Pterin concentrations in the samples were determined in comparison to standard pterins (Schricks, Wettswil, Switzerland). 2.11. Concentration of FeLV p27 antigen The ELISA for quantitative determination of FeLV p27 antigen was developed and described by Lutz et al. (1983). 2.12. Statistical evaluation Statistically significant differences between the Baypamun1 and the placebo groups in all of the parameters monitored were evaluated by Student's t-test (laboratory, immunological, and virological parameters), Mann±Whitney's U-test (clinical para-
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meters), and Chi-square test (conversion to FeLV-negative and survival rate). Statistical significance (p) was fixed at 0.05. 3. Results 3.1. Study I In study I, 120 naturally FeLV-infected cats were treated; 60 cats received Baypamun1, 60 received placebo. Seven of the 60 cats (11.7%) treated with Baypamun and four of the 60 cats (6.7%) receiving placebo converted to a FeLV-negative state during the 6-week therapy interval (Table 2). The difference in the number of converting cats was not statistically significant (p0.343). The mean course of the serum FeLV p27 antigen concentrations during the treatment in study I is shown in Fig. 1. On average the antigen concentration decreased by 7.2% in the cats of the Baypamun1 group and by 5.5% in the placebo group. No significant difference could be shown (p0.851). The general clinical status improved in 47% and 33% of the cats receiving Baypamun1 and placebo, respectively. In the Baypamun1 group, 32% of the cats gained weight, while in the placebo group only 27% increased in body weight. Neither Table 2 Percentage of cats converting to FeLV-negative and mean change of the concentration of FeLV p27 antigen during study I in the cats receiving Baypamun1 and in the cats receiving placebo, and p-value of the differences between the two groups
Conversion to FeLV-negative (%) Concentration of FeLV p27 (%)
Baypamun1
Placebo
p
11.7 ÿ7.2
6.7 ÿ5.5
0.343 0.851
Fig. 1. Mean changes of the serum FeLV p27 antigen concentration during the treatment in study I.
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Fig. 2. Mean changes of the serum FeLV p27 antigen concentration during the treatment in study II.
the differences in the general clinical status (p0.253) nor in the weight change (p0.584) were statistically significant. 3.2. Study II In study II, 30 FeLV-infected cats were treated, 15 cats received Baypamun1, and 15 received placebo. Remission of viremia occurred in two cats of each group (15.4%). The FeLV antigen level was decreased by 1.9% on average after Baypamun1 treatment and increased by 3.7% on average in the placebo recipients (Fig. 2). The difference was not statistically significant (p0.842). Twenty parameters including improvement of lower respiratory tract disorders, rhinitis, stomatitis, weight change, index of Karnofsky, hemoglobin value, PCV, relative and absolute CD4 counts, relative and absolute CD8 counts, CD4/CD8 ratio, neopterin concentration in serum measured by RIA, 7-xanthopterin concentration in serum and urine measured by HPLC, biopterin concentration in serum and urine measured by HPLC, number of cats converting to FeLV-negative, concentration of FeLV p27 antigen, and survival rate were evaluated (Table 3). In none of these parameters could a statistically significant difference could be demonstrated between Baypamun1 and placebo application. 4. Discussion There was no statistically significant difference regarding any of the investigated immunological, clinical, laboratory, or virological parameters between animals receiving Baypamun1 or placebo neither in the first nor second study. In 11 cats of study I and four cats of study II, there was a conversion from a FeLV-positive to a FeLV-negative state but the difference in number of cats was not statistically significant between the groups. These cats were generally younger than 1 year and had no clinical signs. It can be assumed that they were in a state of transient viremia.
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Table 3 Mean changes of the evaluated parameters during study II in the cats receiving Baypamun1 and in the cats receiving placebo, and p-value of the differences between the two groups Baypamun1 Lower respiratory disorders (score) Rhinitis (score) Stomatitis (score) Weight change (kg) Index of Karnofsky (score) Hemoglobin value (g dlÿ1) PCV (%) Relative CD4 count (%) Absolute CD4 count (cells mlÿ1) Relative CD8 count (%) Absolute CD8 count (cells mlÿ1) CD4/CD8 ratio Neopterin in serum (nmol lÿ1) 7-Xanthopterin in serum (nmol lÿ1) 7-Xanthopterin in urine (nmol mmolÿ1)a Biopterin in serum (nmol lÿ1) Biopterin in urine (nmol mmolÿ1)a Conversion to FeLV-negative (%) Concentration of FeLV p27 (%) Survival rate (%)
ÿ0.5 ÿ0.4 ÿ1.5 0.1 8.1 ÿ1.3 ÿ0.1 ÿ3.3 82.2 0.0 39.4 ÿ0.4 ÿ6.6 0.9 0.0 ÿ20.0 ÿ0.1 15.4 ÿ1.9 33.3
Placebo
p
0.1 ÿ0.2 ÿ0.3 0.2 ÿ1.3 1.1 0.1 ÿ1.1 6.1 ÿ0.5 24.6 0.3 ÿ2.5 ÿ0.9 ÿ0.7 1.8 0.0 15.4 3.7 60.0
0.338 0.779 0.481 0.140 0.145 0.851 0.797 0.964 0.810 0.343 0.590 0.744 0.326 0.375 0.100 0.129 0.163 1.000 0.842 0.143
a
7-Xanthopterin and biopterin in urine were measured in nmol mmolÿ1 urine creatinine.
As shown in these studies, there was no cure of FeLV infection by the paramunity inducer Baypamun1. There is no explanation of the divergent results by HoÈrber and Mayr (HoÈrber and Mayr, 1991; HoÈrber et al., 1992; Mayr and HoÈrber, 1992) who stated that they were able to cure 80±100% FeLV-infected cats from viremia by using Baypamun1. Certainly, an accurate diagnosis of FeLV infection is a prerequisite for reliable results. For this reason we considered cats FeLV-positive when the serum was tested positive in three independent ELISA of at least two different samples to avoid inclusion of false-positive animals. Especially in study II, we tried to take a lot of parameters into consideration. It was shown that most of the clinical parameters, the general clinical status, and the quality of life, measured by the index of Karnofsky, improved on average in both groups. This was due to two reasons. Firstly, the cats were presented to the hospital for injections and investigations very often and therefore were under excellent veterinary care during the treatment period. Secondly, supportive management and treatment of secondary infections were provided to all cats. For proper interpretation of the results, comparison with a placebo group is unavoidable. To prevent the subjective influence of the investigator, the study was also double-blind so that objective improvement could be determined. In these two studies, no side-effects of Baypamun1 treatment were observed. However, there was also no detectable influence of Baypamun1 treatment on the FeLV infection. Thus, Baypamun1 treatment cannot be recommended for treatment of FeLV
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infection. The importance of conducting treatment studies as placebo-controlled doubleblind trials was demonstrated in this investigation. Acknowledgements The authors thank Dr. Christian Leutenegger and Dr. Erich Lederer for their advice and help. This research was supported by the Human Capital and Mobility Programme of the European Commission and by Bayer, Leverkusen, Germany. References Baxby, D., 1981. Jenner's Smallpox Vaccine. Morrison and Gibb, London. BuÈttner, M., Mayr, A., 1986. Tests on protection against viral diseases. Comp. Immun. Microbiol. Infect. Dis. 9, 205±215. BuÈttner, M., 1993. Principles of paramunization, option and limits in veterinary medicine. Comp. Immun. Microbiol. Infect. Dis. 18, 1±10. Fuchs, D., Unterweger, B., Hausen, A., Reibnegger, G., Werner, E.R., Hengster, P., Hinterhuber, H., Dierich, M.D., Wachter, H., Blattner, W.A., Weiss, S.H., 1988. Anti-HIV-1 antibodies, anti-HTLV-1 antibodies and neopterin levels in parenteral drug addicts in the Austrian Tyrol. J. Acquir. Immune Defic. Syndr. 1, 65±66. Goldberg, M., Gassner, F., Singer, L.J., Merkenschlager, M., 1989. Pteridinkonzentrationen in Harn und Organgeweben von Hund und Katze bei verschiedenen Neoplasien und Virusinfektionen. TieraÈrztl. Prax. 5, 37±41. Gooding, L.R., 1992. Virus proteins that counteract host immune defenses. Cell 71, 5±7. Hartmann, K., 1989. Referenzbereiche in der Labordiagnostik der Katze. TieraÈrztl. Prax. 5, 58±62. Hartmann, K., 1995. Entwicklung eines Testsystems zur Erprobung neuer Medikamente gegen die FIV-Infektion der Katze als Modell fuÈr die Behandlung erworbener ImmunschwaÈchesyndrome. Habil. med. vet., MuÈnchen. Hartmann, K., Kuffer, M., Kraft, W., 1997. Modifikation des Karnofsky-Indexes fuÈr die Katze zur Beurteilung der LebensqualitaÈt and Therapiekontrolle. TieraÈrztl. Prax. 25, 540±542. HoÈrber, D., Mayr, B., 1991. Paramunisierung FeLV-positiver Katzen mit PIND-AVI. TieraÈrztl. Prax. 19, 311± 314. HoÈrber, D., Schnabl, W., Mayr, B., 1992. Praxiserfahrungen bei der Paramunisierung FeLV-positiver Katzen mit Baypamun HK. TieraÈrztl. Umschau 47, 556±560. Karnofsky, D.A., Abelmann, W.H., Craver, L.F., Burchenal, J.H., 1948. The use of the nitrogen mustard in the palliative treatment of carcinoma. Cancer, 634±656. Lutz, H., Pedersen, N.C., Durbin, R., Theilen, G.H., 1983. Monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27. J. Immuno. Met. 56, 209±220. Mayr, A., BuÈttner, M., 1984. Neue Erkenntnisse uÈber die Grundlagen der ParamunitaÈt und Paramunisierung. Berl. MuÈnch. TieraÈrztl. Wschr. 97, 429±435. Mayr, A., 1982. ParamunitaÈt und Paramunisierung. J. Vet. Med. B 29, 5±23. Mayr, B., HoÈrber, D., 1992. Paramunisierung FeLV-positiver Katzen ± ein Bericht aus der Praxis. Kleintierprax. 37, 515±518. Reddy, M.M., McKinley, G.F., Grieco, M.H., 1991. Evaluation of HIV p24 antigen, beta2-microglobulin, neopterin, soluble CD4, soluble CD8, and soluble interleukin-2 receptor levels in patients with AIDS or AIDS-related complex treated with 20 ,30 -dideoxyinosine (ddI). J. Clin. Lab. Anal. 5, 396±398.