Effects of postirradiation carboxymethylglucan administration in mice

Int..L lmmunopharmac., Vol. 17, No. 3, pp. 167-174, 1995 Elsevier Science Ltd Copyright © 1995 International Society for lmmunopharmacology Printed in Great Britain. All rights reserved 0192-0561/95 $9.50 + .00

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EFFECTS OF POSTIRRADIATION CARBOXYMETHYLGLUCAN ADMINISTRATION IN MICE M. HOFER,* M. POSPi~IL,* ~. VIKLICK~,* I. PIPALOV,/~, * J. HOL,~,* J. NETiKOV,/k * and J. ~ANDULA* *Institute of Biophysics, Academyof Sciences of the Czech Republic, Brno, Czech Republic;and *Instituteof Chemistry, SlovakAcademy of Sciences, Bratislava, Slovak Republic Received 5 August 1994 and in final form 24 November 1994)

Abstract - - The hemopoiesis-enhancingability of a soluble glucan derivative, i.e. carboxymethylglucan(CMG), was investigated in gamma-irradiatedmice. Attention was focused on the usefulness of its single or repeated postirradiation administration.CMG was administeredi.p. at (a) single dose of 6 mg 2 h postirradiation,(b) four 6 mg doses in the first4 dayspostirradiation,(c) four ! .5 nagdoses at the same time intervals.Indicesof granulopoiesis and inflammatoryside effects (liver weight increaseand hepatic granulomas) were investigatedin mice irradiated with a sublethaldose of 7 Gy. ALlthree CMG-treatedgroups of mice were found to exhibit enhancedhemopoietic recovery in comparisonwith the controls. Althoughthe mice repeatedly given the 6 mg CMG doses showed the most rapid recoveries of all the evaluatedparameters of granulopoiesis,the most pronounced hepatic side effects were found in these mice, too. When survivalof mice was recorded in lethally (9 Gy) irradiated animals,the best protective responsewere obtainedfollowingthe repeated administrationof the 1.5 mg CMG dose, the survivalby day 30 in this group being significantlyhigher not only in comparison with the controls but also with the mice repeatedly given the 6 nag dose of CMG. The results suggest that the postirradiation CMG administrationcan be useful for enhancing radiation suppressed hemopoiesis. However, repeated larger CMG doses may produce side effects which compromisethe overall survivalof irradiated mice.

Hemopoietic injury is a critical problem associated with radiation exposure. Therefore, pharmacological support of hemopoiesis damaged by ionizing radiation is of considerable interest. One of the agents which have been tested for their ability to minimize these radiation effects is glucan, a beta-l-3-1inked polyglucose derived from the yeast Saccharomyces cerevisiae. Glucan is a potent stimulant of the cellular and humoral immune systems (DiLuzio, 1983, 1985), and the hemopoietic system (Patchen & MacVittie, 1983). Two distinct forms of glucan have been subject to extensive exploration, i.e. the particulate and the soluble (referred to as gluean-F) forms, prepared and described by DiLuzio et al. (1979). When particulate gluean or gluean-F was administered to mice in a therapeutic regimen 1 h after irradiation, an enhancement ofhemopoietic recovery was observed, though not so expressive when compared with the preirradiation treatment (Pospi~il et al., 1982; Patchen & MacVittie, 1982; Patchen et al., 1984, 1989). Even though both glucan forms were shown to enhance hemopoiesis in irradiated animals, the admini-

stration of the soluble preparation is preferred because of side effects, following the particulate glucan application, which include hepatosplenomegaly and granuloma formation (DiLuzio et al., 1979; Deimann & Fahimi, 1980; Bowers et al., 1986), enhanced susceptibility to endotoxin (Bowers et al., 1986), and microembolization (Patchen et al., 1984). Moreover, postirradiation administration of particulate glucan to lethally irradiated mice impaired survival of the treated animals in comparison with the irradiated control ones (Patchen & MaeVittie, 1985, 1986a). Gluean-F administration generally has not been associated with these deleterious actions, although some observations suggest that even glucan-F treatment is not devoid of some side effects (Williams et al., 1988; Baker et al., 1992). A significant improvement of the state of hemopoiesis was shown in our laboratory in mice treated with a new soluble glucan preparation, carboxymethylglucan (CMG), given alone or in a combination with an inhibitor of prostaglandin production 24 h before single

167

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M. HOFER et al. Table 1. Carboxymethylglucan(CMG) administrationschedule Treatment protocol

2h

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radiation exposures (Pospigil et al., 1991, 1992) or repeatedly in the course of fractionated irradiation (Hofer et al., 1993). Since the postirradiation administration of hemopoiesis-enhancing agents can be of importance in many hazardous situations, the aim of the present study was to define with more precision the possibilities of the postirradiation application of carboxymethylglucan in terms of its curative effects following different single or repeated doses in gamma-irradiated mice. The effects of the treatment were assessed on the basis of the postirradiation recovery of granulopoiesis after sublethal radiation doses, as well as of the survival of mice after lethal radiation exposure. In addition, hepatomegaly, granuloma formation in the liver, and susceptibility of glucan-treated animals to endotoxin were investigated as indices of the possible side effects.

EXPERIMENTAL P R O C E D U R E S

Male (CBAxC57BL)F~ mice, 3 months old, with an average body weight of 22 g, were used. The mice were kept under controlled lighting conditions (L:D 12:12) and temperature was maintained at 22 ± I°C. A standardized pelleted diet and HCl-treated tap water (pH 2-3) were given ad libitum. The mice were exposed to single total-body irradiation from a 6°Co gamma-ray source, at a dose rate of 0.4 Gy/min. During irradiation, the mice were individually placed in chambers in a circular ventilated Plexiglas container. Carboxymethylglucan sodium salt (henceforth referred to as carboxymethylglucan (CMG)), a soluble derivative of glucan, was prepared from the cell walls of the yeast Saccharomyces cerevisiae at the Institute of Chemistry, Slovak Academy of Sciences (Bratislava, Slovak Republic); in the experiments presented, the fraction with a molecular mass of 7.63 Da, estimated by gel permeation chromatography, was used; the degree of substitution with carboxymethyl group per

anhydroglucose unit was 0.91 (Horvfithovfi et al., 1991). CMG was administered after irradiation in different single or repeated doses. Each CMG dose was given intraperitoneally (i.p.) in a volume of 0.2 ml. Saline was used for control i.p. injections. The CMG administration schedule in individual groups of animals is shown in Table 1. Samples of material were taken on days 12, 15, and 18 after 7-Gy irradiation. Blood samples were drawn from a fine incision in the tail vein. For tissue collection, the mice were euthanized by cervical dislocation. Experiments were repeated three times. The numbers of leukocytes and erythrocytes in the peripheral blood, and the cellularity of the femoral bone marrow were determined using a Coulter Counter. Smears of the peripheral blood and of the bone marrow were prepared and stained by the May-Griinwald and Giemsa-Romanowski methods. Relative and, following further calculation, absolute numbers of the evaluated cells were assessed. Bone marrow hemopoietic progenitor cells committed to granulocyte-macrophage differentiation (GM~2FC) were assayed by a semi-solid plasma-clot technique (Vacek et al., 1990). Briefly, femoral bone marrow cell suspensions were plated in quadruplicate using 10% mouse lung-conditionedmedium as a source of colony-stimulating factor. Colonies (>50 cells) were counted after 7 days of incubation in a 37°C humidified environment containing 5% CO2. After determining the wet weight, the livers were embedded in paraffin, the sections stained with hematoxylin-eosin, and granulomas were counted in 15 visual fields at a magnification of 400 ×. Inflammatory lesions, defined as clusters of macrophages, lymphocytes and granulocytes (Yamada et al., 1990) containing more than ten of the above mentioned cells were considered as granulomas. In the experiments using a lethal dose of 9 Gy, the mouse mortality was recorded until day 30 after irradiation. To evaluate whether the CMG treatment in the regimen used modifies the susceptibility of mice to endotoxin, midlethal endotoxin (Salmonella typhimurium, L-6511, Sigma, MO, U.S.A.) dose of 0.16 mg

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12 15 18 DAYS AFTER IRRADIATION Fig. 3. Number of morphologically recognizable cells of the granulocytic lineage in femur in mice irradiated with a dose of 7 Gy. (A) Mice given 6 mg of CMG 1 x, saline 3 x. (B) Mice given 1.5 mg ofCMG 4 x. (C) Mice given 6 mg ofCMG 4 ×. (D) Mice given saline 4 x. Data express means + S.E. from 10-15 measurements, a, b, c, d: the pertinent value is significantly (P<0.05) higher than that of the A, B, C and D group, respectively.

the subtle effects of endotoxin action, the rectal temperature was m e a s u r e d with a t h e r m i s t o r t h e r m o m e t e r within 48 h following 0.12 m g endotoxin administered i.p. in the above given scheme. Monitoring the decrease in core temperature was proposed by Ochalski et al. (1993) for estimating the magnitude o f the endotoxininduced shock-like effects in mice. The statistical s i g n i f i c a n c e o f the d i f f e r e n c e s between arithmetic means was evaluated by a t-test and Dunnett's tables for multiple comparisons with a control (Dunnett, 1964). The significance o f the differences in the survival of the mice was determined by means o f the chi-square test with Yates' correction.

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Fig. 2. Femoral bone marrow cellularity in mice irradiated with a dose of 7 Gy. (A) Mice given 6 mg of CMG 1 x, saline 3 ×. (B) Mice given 1.5 mg of CMG 4 x. (C) Mice given 6 mg of CMG 4 x. (D) Mice given saline 4 x. Data express means 4- S.E. from 10-15 measurements, a, b, c, d: the pertinent value is significantly (P<0.05) higher than that of the A, B, C and D group, respectively.

was administered i.p. to the control and CMG-treated mice at d a y 4 after 9 - G y irradiation, and the 3-day lethality was determined. Furthermore, to investigate

RESULTS

Figures 1-5 illustrate the effects o f C M G treatment on the h e m o p o i e t i c indices a s s a y e d on 12, 15, and 18 days following a 7 Gy radiation dose. The most rapid recovery o f G M - C F C numbers in the bone marrow (Fig. 1) was observed in the group of mice treated repeatedly with the 6 m g dose o f CMG. Only in this group did the G M - C F C counts peak on day 15, whereas in other groups, a continual rise was found. The mean G M - C F C n u m b e r per femur in nonirradiated control mice was 23.6 ± 1.3 x 103.

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Femoral bone marrow cellularity (Fig. 2) was higher in all of the CMG-treated groups when compared with the controls. Nevertheless, only on day 15 after irradiation did all CMG-treated groups exhibit significant

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Fig. 6. Relative liver weight (liver weight/body weight) in mice irradiated with a dose of 7 Gy. (A) Mice given 6 mg of CMG 1 x, saline 3 x. (B) Mice given 1.5 mg ofCMG 4 x. (C) Mice given 6 mg ofCMG 4 x. (D) Mice given saline 4 x. Data express means ± S.E. from 10-15 measurements, a, b, c, d: the pertinent value is significantly (P<0.05) higher than that of the A, B, C and D group, respectively.

differences when compared with controls, with the value o f the group having received 6 mg of CMG repeatedly being the highest. The mean bone marrow cellularity in non-irradiated control mice was 25.1 ± 2.0 x 106. F i g u r e 3 shows the absolute numbers of the morphologically recognizable cells of the granulocytic lineage in the bone marrow of the femur. The highest recovery rate o f these cells by day 18 postirradiation was found in the mice treated repeatedly with the 6 mg C M G dose. The mean number o f the granulocytic lineage cells in the femoral bone marrow in nonirradiated control mice was 15.7 ± 1.6 x 10 6. The differential counting of white blood cells in the b l o o d r e v e a l e d preferential effects of the CMG administration on granulocytes (Fig. 4). While, in this case, the values o f all CMG-treated groups were significantly higher in all time intervals studied when c o m p a r e d with the control group, the lymphocytes s h o w e d no significant enhancement (Fig. 5). The mean numbers o f granulocytes and lymphocytes in the b l o o d in non-irradiated control mice were 1.02 ± 0.09 x 10Vial, and 5.43 ± 0.41 x 10VI~I, respectively. The relative liver weight (liver weight/body weight) (Fig. 6) was found to be higher in all CMG-treated groups o f mice in comparison with the controls in all time intervals studied. The mice which had received the dose o f 6 mg o f CMG repeatedly exhibited the highest relative liver weights. On days 15 and 18 after

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irradiation, the values in mice which had received the dose of 6 mg of CMG either singly or repeatedly were significantly higher in comparison with the controls; moreover, on day 18, a statistical significance was also found when comparing relative liver weights between the mice administered 6 mg CMG dose singly or repeatedly and the mice repeatedly given a lower (1.5 mg) dose. The relative liver weight in non-irradiated control mice was 0.036 + 0.001. The histological liver examination performed on day 15 following the sublethal irradiation has revealed a high number of hepatic granulomas in the mice treated with the repeated doses of 6 mg of CMG (Fig. 7). The granulomas were more numerous in this group in comparison with both the control mice and the mice from the other CMG-treated groups. A separate experiment was performed to examine whether the pathological changes in the liver were caused solely by CMG or if irradiation also contributed to these effects in CMG-treated mice. The liver weights and the number of hepatic granulomas were ascertained both in irradiated (7 Gy) and sham-irradiated mice given either four times 6 mg of CMG or four times saline in the time intervals as mentioned above. In both the irradiated and sham-irradiated animals, the liver weight and the number of hepatic granuiomas were significantly higher in the CMG-treated mice in comparison with the saline-treated ones. No significant differences were found, either between irradiated and sham-irradiated

Fig. 8. Survivalof mice irradiatedwith a dose of 9 Gy. (k) Mice given 6 mg ofCMG 1 ×, saline 3 ×. (B) Mice given 1.5 mg of CMG 4 x (C) Mice given 6 mg of CMG 4 ×. (D) Mice given saline 4 ×. Thirty animals per group were used. Data were obtainedfrom three replicateexperiments,a, b, c, d: the pertinent value is significantly (P<0.05) higher than that of the A, B, C and D group, respectively. CMG-treated animals or between irradiated and shamirradiated saline-treated mice (results not shown). Following the irradiation with the lethal dose of 9 Gy, the survival of the mice treated four times with the 1.5 mg dose of CMG was found to be the highest one (50% by day 30) and significantly different in comparison not only with the control group (13%) but also with the mice treated repeatedly with the 6 mg CMG dose (23%) (Fig. 8). Experiments evaluating the susceptibility of CMGtreated mice to endotoxin did not yield positive results. There were no differences in endotoxin sensitivity in terms of lethality or a hypothermic response among the CMG-treated and control groups of mice challenged with endotoxin on day 4 after the lethal dose of 9 Gy (data not shown).

DISCUSSION

The macrophage activating potency of glucan (DiLuzio, 1983, 1985) and the effects of glucan on hemopoiesis are probably inter-related events. It was shown that augmented granulopoiesis after particulate glucan is probably mediated by an increased release of granulocyte-macrophage colony-stimulating activity (GM-CSA) from macrophages (Burgaleta & Golde, 1977; Niskanen et al., 1978). Increased serum GM-CSA levels have also been observed in mice receiving watersoluble glucans, i.e. glucan-F (Patchen & MacVittie, 1986b) and carboxymethylglucan (Hofer et al., 1993;

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Pospi~il et al., 1993). For this reason, our investigations were focused on the granulocyte production, and included an assessment of the bone marrow compartment o f stem cells committed to granulocyte and/or macrophage development (GM-CFC), morphologically recognizable cells of the granulocytic lineage in the bone marrow, and granulocytes in the peripheral blood. It is well known that granulocytopenia is a major factor contributing to infection-induced morbidity and mortality after radiation exposure (Walker, 1978). Glucan action may be accompanied by detrimental side effects. In association with the proliferative response of the macrophage system, large populations of inflammation-mediating elements appear in the liver resulting in hepatomegaly and granuloma formation (DiLuzio et al., 1979; Deimann & Fahimi, 1980; Bowers et al., 1986; Yamada et al., 1990; Way et al., 1985). It was suggested that these effects were produced by the particulate glucan and not with its soluble derivative, glucan-F (DiLuzio et al., 1979; Bowers et al., 1986). However, prolonged treatment of mice for a period of 60 days with glucan-F at doses of 1000 mg/kg produced granuloma formation in the liver (Williams et al., 1988). Recently, Baker et al. (1992) described transient hepatomegaly and associated histologic changes in mice treated with a single dose (200 mg/kg) of glucan-F. Furthermore, particulate forms of glucan have been reported to enhance the susceptibility of animals to endotoxin (Bowers et al., 1986; Cook et al., 1980; Lazar & Agarwal, 1982). Both granuloma formation and endotoxin sensitivity seem to be linked with the enhanced activity of the retieuloendothelial system and increased release of a variety o f inflammatory mediators. Experiments of Way et al. (1985) and of Bowers et al. (1986) have suggested the potential role of arachidonic acid metabolites in mediating the hepatic inflammatory response to particulate glucan. Also, interleukin-1 and tumor necrosis factor-alpha, monokines, which are known to be released by glucan action (Sherwood et al., 1987; Abel & Czop, 1992), have been found to play an essential role in granuloma formation (Kasahara et al., 1989). The potential impact of glucan's side effects should be taken into account when using this biological response modifier for treatment of the hemopoietic radiation damage. One consequence of granuloma formation in the liver, and of the released cytotoxic inflammatory mediators from macrophages, such as damaging oxygen radicals, proteolytic enzymes, and aracbidonate metabolites (Bowers et al., 1986; Walker, 1978) could be hepatic dysfunction. For example, in the experiments of Bowers et al. (1986), particulate glucan-treated rats exhibited altered glucose metabolism and had become hypoglycemic by 4 h after exposure to

endotoxin. According to Walker (1978), a postirradiation escape of endotoxin from the gut and subsequent endotoxemia are important pathogenic mechanisms of the lethal radiation syndrome. Thus, glucan-induced increased sensitivity to endogenous endotoxin might aggravate the damage associated with endotoxin toxicity to many tissues, as well as with systemic effects of endotoxin-induced circulatory collapse. In order to ascertain the possible participation of the undesirable effects of glucan, our experimental protocol included the investigations of hepatic granuloma formation and of endotoxin sensitivity of CMG-treated mice. Our results suggested that the postirradiation CMG treatment was effective in enhancing granulopoiesis in sublethally irradiated mice. Not only preirradiation, i.e. "protective" treatment shown earlier (Pospigil et al., 1991, 1992), but also postirradiation, i.e. "therapeutic" treatment with CMG was beneficial. The stimulatory responses seemed to be heightened by the repeated administration of the drug (see comparison of the groups given 6 mg dose once and four times), as well as by the elevation of the administered dose (see comparison of the groups given 1.5 mg and 6 mg doses repeatedly). Thus, the repeated administration of CMG can be effective and exhibits a dose dependence, at least in the treatment regimen used. Our data indicated that the soluble derivative of glucan used in our experiments was not devoid of side effects, as shown by the evaluation of the hepatic response. This is in accordance with the two findings mentioned above (Williams et al., 1988; Baker et al., 1992) using a soluble glucan form termed glucan-F. Like the hemopoiesis stimulating effects, the hepatic side effects exhibited a dose-dependence, although the hepatic granuloma formation seemed to increase more steeply at the higher CMG dosage as compared with the hemopoietic effect. The overall lethal response of the lethally irradiated and CMG-treated mice may be the result of the balance between the positive effects of the treatment with CMG on hemopoiesis and its undesirable side effects. Even though the hemopoietic damage is the major cause of death with a dose of 9 Gy, complexity of pathophysiological events of radiation syndrome including inflammatory reactions and metabolic disorders has to be taken into account. It was shown that an exaggerated inflammatory reaction could play an unfavourable role in the radiation syndrome (DeGowin & Lass, 1985). Our experiments evaluating the survival of lethally irradiated mice treated with CMG support such a contention. The survival enhancing effects of CMG were lower when repeatedly using the 6 mg dose as compared with the 1.5 mg dose. Thus, it is possible that unfavourable side effects of CMG counteract the benefit of hemopoietic stimulation. The

Postirradiation Carboxymethylglucan Administration in Mice pathogenic mechanisms of these unfavourable effects of C M G remain to be further analyzed. In conclusion, our results suggest that the soluble form o f glucan, i.e. c a r b o x y m e t h y l g l u c a n , c o u l d be useful for the p o s t i r r a d i a t i o n t r e a t m e n t o f t h e hemopoietic damage. However, attention should be paid to the optimal dosing of this hemostimulatory agent.

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Acknowledgements - - The authors wish to thank Ms V. Reichmannov~i for her skilful technical assistance. This work was supported by a grant (604104) from the Academy of Sciences of the Czech Republic. The research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Sciences.

REFERENCES Abel, G. & Czop, J. K. (1992). Stimulation of human monocyte beta-glucan receptors by glucan particles induces production of TNF-alpha and IL-lbeta. Int. J. Immunopharmac., 9, 1363-1373. Baker, W. H., Nold, J. B., Patchen, M. L. & Jackson, W. E. (1992). Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HEN mice. Proc. Soc. exp. biol. Med., 201,180---191. Bowers, G. J., Patchen, M. L., MacVittie, T. J., Hirsch, E. F. & Fink, M. P. (1986). A comparative evaluation of particulate and soluble glucan in an endotoxin model. Int. J. lmmunopharmac., 8, 313-321. Burgaleta, C. & Golde, D. W. (1977). Effect of glucan on granulopoiesis and macrophage genesis in mice. Cancer Res., 37, 1739-1742. Cook, J. A., Dougherty, W. J. & Holt, T. M. (1980). Enhanced sensitivity to endotoxin induced by the RE stimulant, glucan. Circ. Shock, 7, 225-238. DeGowin, R. L. & Lass, S. U (1985). Chronic inflammation impairs hematopoiesis and survival after irradiation. 3". Lab. clin. Med., 105, 299-304. Deimann, W. & Fahimi, H. D. (1980). Hepatic granulomas induced by glucan. An ultrastructural and peroxide-cytochemical study. Lab. Invest., 43, 172-181. DiLuzio, N. R. (1983). Immunopharmacology ofglucan: a broad spectrum enhancer of host defense mechanisms. Trends Pharmac. Sci., 4,344--347. DiLuzio, N. R. (1985). Update on the immunomodulating activities of glucans. Springer Semin. lmmunopath., g, 387-400. DiLuzio, N. R., Williams, D. L., McNamee, R. B., Edwards, B. F. & Kitahama, A. (1979). Comparative tumor-inhibitory and antibacterial activity of soluble and particulate glucan. Int. J. Cancer, 24, 773-779. Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, 482-491. Hofer, M., Pospigil, M., ViklickL ~., Vacek, A., Pipalov~t, I. & Bartoni~kovL A. (1993). Hematopoietic recovery in repeatedly irradiated mice can be enhanced by a repeatedly administered combination of diclofenac and glucan. J. Leukoc. Biol., 53, 185-189. Horv~.thov~i,M., ~oltrs, L., Lut6nsk~., H., ~;andula, J. & Mislovi~,ov/t, D. ( 1991). Study of water soluble beta- 1,3-glucan denaturation with dimethylsulfoxide using gel permeation chromatography. J. appl. Polym. Sci., 48, 33-38. Kasahara, K., Kobayashi, K., Shikama, Y., Yoneya, 1., Kaga, S., Hashimoto, M., Odagiri, T., Soejima, T., Ide, H., Takahashi, T. & Yoshida, T. (1989). The role of monokines in granuloma formation in mice: the ability of interleukin-1 and tumor necrosis factor-alpha to induce lung granulomas. Clin. Immun. lmmunopath., 51, 419--425. Lazar. G. & Agarwal, M. K. (1982). Additive effects of glucan and streptozotocin on endotoxicosis in mice. Med. Microb. lmmun., 171, 179--186. Niskanen, E. O., Burgaleta, C., Cline, M. J. & Golde, D. W. (1978). Effect of glucan, a macrophage activator, on murine hemopoietic cell proliferation in diffusion chambers in mice. Cancer Res., 38, 1406-1409. Ochalski, S. J., Hartman, D. A., Belfast, M. T., Walter, T. L., Glaser, K. B. & Carlson, R. P. (1993). Inhibition of endotoxininduced hypothermia and serum TNF-alpha levels in CD-I mice by various pharmacological agents. Agents Actions, 39, C52-C54. Patchen, M. L. & MacVittie, T. J. (1982). Use of glucan to enhance hemopoietic recovery after exposure to cobalt-60 irradiation. In Macrophages and Natural Killer Cells (eds Normann, S. J. and Sorkin, J.), pp. 267-272. Plenum Press, New York. Patchen M. L. & MacVittie, T. J. (1983). Dose-dependent responses of murine pluripotent stem cells and myeloid and erythroid progenitor cells following administration of the immunomodulating agent glucan. Immunopharmacology, 5, 303-313. Patchen M. U & MacVittie, T. J. (1985). Stimulated hemopoiesis and enhanced survival following glucan treatment in sublethally and lethally irradiated mice. Int. J. Immunopharmac., 7,923-932. Patchen M. L. & MacVittie, T. J. (1986a). Comparative effects of soluble and particulate glucans on survival in irradiated mice. J. biol. resp. Modif., 5, 45-60. Patchen M. U & MacVinie, T. J. (1986b). Hemopoietic effects of intravenous soluble glucan administration. J. lmmunopharmac., 8, 407-425. Patchen M. L., MacVittie, T. J. & Jackson, W. E. (1989). Postirradiation glucan administration enhances the radioprotective

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