Administration to mice of a low, non-immunosuppressive dose of cyclophosphamide 4 days before infection with foot-and-mouth disease virus decreases viral replication, enhances the immune response against the virus and prevents pancreatic damage.Facultad de Ciencias Médica

Di Girolamo, María Teresa Vanda

Laguens, Rubén Martín

Portiansky, Enrique Leo

Cellular and Molecular Life Sciences

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110 Experienfia 45 (1989), Birkhfiuser Verlag, CH-4010 Basel/Switzerland Short Communications Protective and immunostimulating activity of a low dose of cyclophosphamide in the experimental infec- tion of mice with foot-and-mouth disease virus E. L. Portiansky, W. M. T. DiGirolamo and R. P. Laguens Chtedra de Patologia 11, Facultad de Ciencias M6dicas, Universidad Nacional de La Plata, 1900 La Plata (Argentina) Received 16 May 1988; accepted 5 October 1988 Summary. Administration to mice of a low, non-immunosuppressive dose of cyclophosphamide 4 days before infection with foot-and-mouth disease virus decreases viral replication, enhances the immune response against the virus and prevents pancreatic damage. Key words. Foot-and-mouth disease virus; cyclophosphamide; pancreatitis; immune response. The immunosuppressive activity of  cyclophosphamide (Cy) is currently used to explore the putative immune pathogene- sis of tissue damage in viral infections 1 - 3, It is usually held that if lethality or tissue alterations are prevented or de- creased by treatment with the drug immune mechanisms would probably be involved in the production of alter- ations 3. However, when it is administered at a low dose and before antigenic challenge, Cy can display an immunomodu- lating activity, enhancing antibody synthesis 4. On this basis we decided to study the effect of a low dose of this drug on the evolution of the experimental infection of mice with foot- and-mouth disease virus (FMDV). We present herein evidence that when a low dose of Cy is administered before infection with FMDV, viral replication and tissue damage can be prevented and the immune re- sponse against the virus is enhanced. Materials and methods. Experimental design: Adult 3-month- old male and female outbred Swiss mice were infected by intraperitoneal route (i.p.) with 1 x 104, 5 x 104 or 1 x 105 PFU of F M D u  strain 029. Cyclophosphamide (50 mg/kg) was administered i.p. 4 days before infection. Groups of 3 animals each were sacrificed at 1,2,3,7,21,28,60 and 90 days post-infection (p.i.). Pancreas and blood were collected for virus isolation and for searching of antibodies against FMDV. Controls were animals infected with FMDV but not pulsed with Cy. Virological studies: The 029 strain FMDV grown in BHK 21 monolayers was used for all the experiments. Virus concen- tration in supernatants of  BHK 21 infected cells and from blood or pancreas was determined by the plaque-forming assay on methyl-cellulose covered BHK 21 monolayers. For quantitation of FMDV in pancreas the organ was homoge- nized in MEM Eagle's (1/10 W/V), Suspensions were cen- trifuged at 25,000 g for 30 min at 4 ~ and tenfold dilutions of the supernatant was absorbed on BHK 21 monolayers for 1 h at 37 ~ Titer was expressed as PFU/ml for samples of BHK21 supernatants and blood or PFU/g for samples of pancreas. Neutralization assay: Serum was separated from the blood samples and kept at - 7 0  ~ Neutralizing activity against FMDV was assayed by the plaque-forming test. Titer was expressed as the reciprocal of a base 2 maximal dilution which gave a 80 % reduction of  the number of  plaques on BHK21 monolayers. Immune-complexes detection: In order to determine the pres- ence of immune-complexes in the sera of the Cy-treated and non-treated mice the polyethyleneglycol (PEG) precipitation technique developed by Creighton et al. 5 was carried out. Briefly, serum (1 ml diluted l/10 in borate buffer) was mixed with 1 ml of 7 % PEG in borate buffer, pH 8.6 and incubated at 4 0C for 18 h. The mixture was then centrifuged at 20,000 g for 30 min at 4 ~ The supernatant was discarded and the precipitate was washed, centrifuged with 2 ml of 3.5 % PEG in borate buffer at 20,000 g for 30 rain at 4 ~ resuspended in 2 ml of 0.1 M NaOH and read in a spectrophotometer at 280 nm. The statistical analysis of the results was done ac- cording to Fisher s method 6. Histopathological studies': Samples of  pancreas, spleen, liver, tongue, heart, lungs and kidney were fixed in Bouin's fluid, embedded in paraffin, sectioned at 7 gm and stained with hematoxylin and eosin. Results. Histopathologicalfindings: The only lesion found in the infected mice not treated with Cy was a marked pancre- atic necrosis with polymorphonuclear cell infiltrates. Lesions were evident at 24 h p.i. with 1 x 104 PFU,  when small foci of acinar cells showed pyknosis and cytoplasmic eosinophilia with disappearance of secretory granules. Polymorphonucle- ar margination in the small blood vessels and a marked inter- stitial edema were also present at that time. Between 48 h and 72 h p.i. most of the acini appeared necrotic, and the interstitial space was infiltrated with polymorphonuclear leukocytes (fig. 1). At 4 days p.i. lesions were less intense and by day 5 p.i. no inflammatory infiltrates were present and numerous mitoses were seen in the acini. At 7 days p.i. the pancreas looked normal. In no case were the islets of Langer- hans damaged. In mice treated with a low dose of Cy 4 days before infection lesions were ahnost absent. Only a few acini appeared ne- crotic at 48-72 h p.i. but the marked intercellular edema and the polymorphonuclear inflammatory infiltrate were not ev- ident at any time p.i. (fig. 2). In the remnant organs (e.g. spleen, liver, tongue, heart and kidney) no lesions were observed in Cy-treated and non- treated animals at any time p.i. No differences were observed when animals were inoculated with higher amounts of FMDV. Virological studies: In the infected animals not treated with Cy virus could be recovered from pancreas as early as at 12 h p.i. Virus titer increased steadily until 24 h p.i. and decreased at 48 h p.i. At day 3 p.i, no virus could be recovered. In Figure 1. Pancreas of a mouse killed 48 h after infection with FMDV. Polymorphonuclear infiltrates, edema and acinar necrosis are evident. Hematoxylin and eosin, x 250. Short Communications Experientia 45 / Figure 2. At 48 h after'receiving FMDV the pancreas of a mouse treated with Cy 4 days before infection shows an almost normal appearance. Hematoxylin and eosin, x 250. blood,  virus was detected at  24 h p.i. and  its concen t ra t ion  increased unti l  48 h. At  day 3 p.i. no  virus could be recovered f rom blood. In animals  pre t rea ted  with a low dose of  Cy virus yields f rom pancreas  was similar to the cont ro l  non- t rea ted  group at 12 h p.i.; at  24 h p.i. virus concen t ra t ion  was more  than  1 log lower than  the cont ro l  and  at 48 h p.i. no virus could be recovered. In bo th  groups,  circulat ing virus was detected in b lood  at 24 48 h p.i. A l though  virus titer was lower in the Cy-treated animals,  the difference between bo th  groups was not  significant. Results  are summarized  in tables 1 and  2. At t empts  to recover virus f rom the o ther  organs  were nega- tive at  any time p.i. Anti-FMDV immune response. In the cont ro l  Cy non- t rea ted  mice neutral izing ant ibodies  in serum could be detected as early as 3 days p.i. Figure 3 shows tha t  m a x i m u m  titer was present  at  day 21 and  decreased at day 28. No neutral iz ing ant ibodies  were detected in the sera collected at days 60 and  90 p.i. In the animals  injected with Cy, a several-fold higher  titer of  neutra l iz ing ant ibodies ,  as compared  with the non- t rea ted  (1989), Birkh/iuser Verlag, CH-4010 Basel/Switzerland 111 group was present  f rom day 3 p.i. and  remained high until  day 28 p.i. A t  60 and  90 days p.i., when  no ant ibodies  could be detected in the group not  t reated with Cy, titers as h igh as / /640 and  1/320, respectively, were still present.  Previous  exper iments  had  shown tha t  the same titer of  neutra l iz ing ant ibodies  were ob ta ined  until  day 30 p.i. with different  dos- es of  F M D V .  Immune-complexes detection: In all cases, the concen t ra t ions  of  immune-complexes  in the sera of  Cy-treated and  non-  t reated mice were similar. No  significant differences were found  between b o t h  groups  at  any t ime p.i. Discussion. F o o t  and  m o u t h  disease is a major ,  a l though  rarely fatal  disease o f  cattle and  other  domest ic  animals  7. Infect ion of  adul t  mice with F M D V  produces  a self-limiting disease wi th  no  mortal i ty .  The  only lesion found  is a severe pancrea t ic  necrosis,  es tabl ished as early as 24 h p.i. 8, associ- ated with a p o l y m o r p h o n u c l e a r  i n f l ammato ry  infiltrate.  Vi- rus replicates in the pancreas  unti l  day 2 p.i. an d  a mild viremia is also observed.  Nei ther  histological  lesions, no r  virus replicat ion can be detected in o ther  organs. The  pancre-  atic lesion and  viral repl icat ion are ephemeral ,  and  by day 7 p.i. the pancreas  regains its no rma l  appearance  and  no virus can be isolated. This is associated with the synthesis of  anti-  Table 1. Virus yield from the pancreas in cyclophosphamide-treated and non-treated mice infected with FMDV. Viral titer was expressed as PFU/g tissue. NVD: no virus detected. Hours p.i. Cy-treated Non-treated 12 2.6 4- 0.10x 103 5 + 0.15 x 103 24 0.9 4- 0,05 x 10 ~ 1.6 4- 0.05 x 104 48 NVD 3 + 0.45 x 103 72 NVD NVD Table 2. Virus yield from blood in cyclophosphamide-treated and non- treated mice infected with FMDV. Viral titer was expressed as PFU/ml. NVD: no virus detected. Hours p.i. Cy-treated Non-treated 12 NVD NVD 24 1.5 + 0,04x 103 6.5 + 0.13 x 103 48 1.3 + 0.05 x 103 5.5 + 0.09 • 103 72 NVD NVD 256o "N 128o 4a - , , 4  ,a  6 4 0  '~ 320 -,-I r, m 160 80 .-- __,I ,. / ,L...,. ~ / I , .  II "I II 3 7 21 28 60 90 days p.i. Figure 3. Titer of anti-FMDV neutralizing antibodies from non-treated (O----O) and Cy-treated (e---e) mice. 112 Experientia 45 (1989), Birkh/iuser Verlag, CH-4010 Basel/Switzerland Short Communications FMDV neutralizing antibodies which attain maximum titers in serum at about 3 weeks p.i. Our results show that pretreatment of mice 4 days before infection with a low dose of Cy prevents the appearance of pancreatic lesions and decreases the virus yield. This was associated with an enhanced synthesis of anti-FMDV neu- tralizing antibodies, which not only reached titers in serum higher than those present in control mice, but also persisted for a longer time. This effect could be tentatively ascribed to the immunomodulating activity of Cy 4. Given at a large dose Cy is a potent immunosuppressor agent 9 - 11. However, it is known that Cy can also display an immunostimulating activity, probably by its action on the suppressor cascade a2 Several reports claim that Cy, administered before antigenic challenge and in a low non-immunosuppressive dose, can enhance antibody formation 13. In other systems, such as in implanted experimental tumors, a low dose of Cy can induce rejection and eventual cures, even when administered at a time where the tumors grow to a considerable size 4. This phenomenon has also been associ- ated to the activity of the drug on the T-suppressor cells 14. Our results show that in Cy-treated mice the synthesis of anti-viral antibodies was markedly enhanced. This phe- nomenon could be responsible for the decrease in virus pro- duction in the pancreas and mildness of pathological alter- ations. Although the lower titer of neutralizing antibody in the serum of infected mice not treated with Cy could be ascribed to the presence of virus-antivirus complexes, this possibility seems unlikely since the level of circulating im- mune-complexes was similar both in the Cy-treated and non- treated mice. The possibility that the drug can display a direct anti-viral effect seems also unlikely. The drug was administered 4 days before infection and according to its short half-life 15, a very small amount of Cy would be present at the time when virus replicates in the pancreas. Moreover, the fact that the first cycle of virus replication was not affected (at 12 h p.i. virus yields from pancreas of Cy-treated and non-treated mice were similar) supports the opinion that Cy, in the dose and schedule of administration we used, has no direct effect on virus replication. Another alternative possibility for explaining the lack of pancreatic alterations is the well-known cytocidal effect of Cy on polymorphonuclear leukocytes a 6. Although leukope- nia could be responsible for the absence of inflammatory infiltrates in the pancreas, other phenomena such as the aci- nar necrosis, which appears earlier than the polymorphonu- clear infiltrate and the diminished virus replication, are un- likely to be related to the decrease in the leukocyte number. 1 Gershwin, M.E., G0etzl, E.J., and Steimberger, A. D., Am. Int. Med. 80 (1974) 531. 2 Scott, D. G. I., and Bacon, P. A., Am. J. Med. 76 (1984) 377. 3 Natanson, N., Monjan, A., Panitch, S. H., Johnson, E., Petursson, G., and Cole, G. A., in: Viral Immunology and Immunopathology, p. 357. Ed. A: L. Notkins. Academic Press, New York 1975. 4 Dray, S., and Mokyr, M. B., in: Immunomodulation. New Frontiers and Advances, p. 349. Eds H. H. Fundemberg, H. D. Whitten and F. Ambrogi. Plenum Press, New York 1984. 5 Creighton, W. D., Lambert, P. H., and Miescher, P. A., J. Immun. 111 (1973) 1219. 6 Snedecor, G. W., in: M&odos estadisticos aplicados a la investigaci6n agricola y biolrgica, p. 91. Compafiia Editorial Continental, Mrxico 1964. 7 Brown, F., Ann. Inst. Pasteur/Virol. 136E (1985) 547. 8 Plan, H., Virology 9 (1959) 484. 9 Fass, L., and Fefer, A., J. Immun. 109 (1972) 749. 10 Lubet, R. A., and Carlson, D. E., J. natl Cancer. Inst. 61 (1978) 897. 11 Turk, J. L., Parker, D., and Poulter, L. W., Immunology 23 (1972) 493. 12 Glaser, M., J. exp. Med. 1499 (1979) 774. 13 Kaufman, S. H. E., Hahn, H., and Diamanstein, T., J. Immun. 125 (1980) 1104. 14 Bollinghoff, M., Starzinski-Powitz, A., Pfizenmaier, K., and Wagne, H., J. exp. Med. 145 (1977) 455. 15 Graul, E. M., Schaumloffel, E., Hundeshagen, H., Williams, H., and Simon, G., Cancer 20 (1967) 896. 16 Van Bekkum, D., Sem. Hemat. 21 (1984) 81. 0014-4754/89/010110-0351.50 + 0.20/0 9 Birkh/iuser Verlag Basel, 1989 Effect of consumption of green and black tea on the level of various enzymes in rats M. Ali*, L. H. J. Hayat, J. A1 Saleh and C. J. Gubler + Department o f  Biochemistry, Faculty o f  Science, Kuwait University, P.O. Box 5969, 13060 Safat (Kuwait) Received 16 June 1988; accepted 13 September 1988 Summary. Drinking of both green and black tea as the only liquid ingested resulted in significant decreases in the activity of transketolase in whole blood of rats both before and after the in vitro addition of thiamin diphosphate. Liver transketolase activity was decreased only by green tea. Mucosal transketolase activity was not affected by either type of tea. The activity of lactate dehydrogenase (LDH) was not affected by either type of tea, while whole blood LDH was decreased by both green and black tea. Neither tea had any affect on mucosal alkaline phosphatase, but thiamin diphosphatase activity was decreased by both teas. An increase in liver total thiamin resulted from the drinking of both types of tea. Key words. Green tea; black tea; transketolase; lactate dehydrogenase. Tea, both green (unprocessed) and black (processed), has been consumed as a beverage from time immemorial and in some societies fermented leaves are chewed as a stimulant. In some cultures, tea is the common drink in place of water. Many plants of nutritional value, including tea, have been shown to possess antithiamin activity a-4. Somogyi and coworkers 5.6 showed that the antithiamin activity was chiefly due to polyhydroxy phenols in the products. Tea is rich in such compounds, notably tannic acid, and has been shown to possess considerable antithiamin activity 3, v. Hence stud- ies have been made on the effects of tea consumption on thiamin nutrition and status in humans 8-1o. Heavy con- sumption of tea was shown to lead to a decrease in thiamin excretion in the urine and to an increase in the percent in- crease in the red cell transketolase activity after the in vitro addition of thiamin diphosphate (%TPP effect), which is considered diagnostic of thiamin deficiency if it is above 20%. 

Protective and immunostimulating activity of a low dose of cyclophosphamide in the experimental infection of mice with foot-and-mouth disease virus

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Protective and immunostimulating activity of a low dose of cyclophosphamide in the experimental infection of mice with foot-and-mouth disease virus

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