Stabilization of Salmonella-specific dialyzable leukocyte extracts

Veterinary Immunology and Immunopathology, 32 (1992) 103-112

103

Elsevier Science Publishers B.V., Amsterdam

Stabilization of Salmonella-specific dialyzable leukocyte extracts I. Mikula, J. Pistl and J. Rosocha Department of Microbiology, Immunology and Animal Hygiene, Universityof Veterinary Medicine, Kosice 041 81, Czechoslovakia (Accepted 2 May 1991 )

ABSTRACT Mikula, I., Pistl, J. and Rosocha, J., 1992. Stabilization of Salmonella-specific dialyzable leukocyte extracts. Vet. Immunol. lmmunopathol., 32:103-112. Activity of Salmonella-specific dialyzable leukocyte extracts (DLE) prepared from mesenteric lymphatic nodes of calves and stabilized with bovine albumin was studied in this work. The effect of ambient temperature and storage period on the activity of DLE was evaluated. Testing for DLE activity by means of capillary leukocyte migration inhibition (LMI) assay showed that DLE stabilized with albumin retained 60% of its activity for 12 months of storage at 4 °C. This level of activity was retained in the native DLE (without albumin) kept at -200C. DLE stabilized with albumin and stored for 12 months at 4 °C inhibited the penetration of salmonellae into the liver and spleen, and their colonization in the gastrointestinal tract was significantly reduced. ABBREVIATIONS BEM, basal Eagle medium; DLE, dialyzable leukocyte extracts; LMI, leukocyte migration inhibition; MID, minimal inhibition dose.

INTRODUCTION

Dialyzable leukocyte extract can induce protection against microbial pathogens. The activity of DLE was tested against numerous viral (Mayer et al., 1985; Wilson and Fort, 1987; Zailian, 1987), bacterial (Buchwald et al., 1987; Mikula and Pistl, 1989 ), protozoan and parasitic diseases (Klesius, 1987 ). The number of different substances in DLE is large, as one might expect, given that the source is the whole cell, and that DLE will include everything that is small (e.g. nucleotides, purine and pyrimidine bases, amino acids, thymosine). This extract contains more than 200 distinct moieties, a number of which have been identified, e.g. thymosin (Burger et al., 1983; Wilson et al., 1983; Huo et al., 1987). The low-molecular and immunologically active DLE compounds are heat© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

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I. MIKULA ET AL.

labile and their biological activity remains unchanged for a period of several months only in the temperature range of - 2 0 to - 70 °C (Wilson et al., 1976; Fudenberg, 1987). Presently, available DLE preparations are stabilized by deep freezing until use (Pek~irek and Cech, 1986). Our work was aimed at stabilization of biological activity of Salmonella-specific DLE by means of 10% apyretic solution of bovine albumin (Mikula et al., 1989). Mobarhan (1988) and Remy and Piznansky (1978) reported on the role of albumin in the transport of drugs and nutrients and also in substitutive enzyme therapy. MATERIALS AND METHODS

Preparation of DLE Salmonella-specific bovine DLE (DLE s-inf) was prepared from mesenteric lymph nodes and spleens of calves immunized with Salmonella vaccine (Salinvak, Bioveta, Nitra, Czechoslovakia) and subsequently infected with the Salmonella typhimurium 4/5 strain as described in another paper (Mikula et al., 1990). The main preparation procedures are the same as those reported by Lawrence ( 1955 ). Bovine lymph nodes and spleens were cleaned, homogenized by the addition of pyrogen-free distilled water in a 2: 1 ratio, then by freezing and thawing, the tissue suspension was centrifuged (at 5000×g for 30 min) and the supernatant was filtered through a whirling asbestic-cellulose filter (Seitz K-3 ). The filtrate was dialyzed against pyrogen-free distilled water containing 0.05% maltose. Dialysis lasted for 72 h, with replacement of distilled HzO every 24 h. The dialyzate was concentrated by freeze-drying, and 100 g of cleaned lymphatic nodes and spleens subjected to dialysis provided, on average, 650 mg oflyophylisate. One gram oflyophylisate was resuspended in 100 ml distilled water and then purified through Amicon apparatus to ensure the molecular weight was up to 10 000. The specified DLE concentration, expressed in optical density units, corresponded, on average, to the value 15 at 260 nm (OD260-- 15 ) and 8 at 280 nm (OD28o= 8 ). The obtained preparation corresponded to qualitative standards according to Akashi et al. ( 1983 ). The content of peptides was determined by the Kjeldahl method (Davidek et al., 1981 ), and ribose content by the orcinol method (Davidek et al., 1981 ). DLE preparations obtained from other sources were also tested: from fattening bulls immunized by the Salinvak vaccine (DLE s- ira); from non-immunized fattening bulls (DLE n). These preparations were used in the clinical experiments in calves (Mikula et al., 1992 ) and were prepared in a similar way to DLE s-inf.

SDS/PAGE Polyacrylamide gel electrophoresis in the presence of SDS was performed by the method utilized in Laemli (1970). A low molecular weight (LMW)

SALMONELLA-SPECIFICDIALYZABLELEUKOCYTE EXTRACTS

105

calibration kit (Pharmacia, LKB Biotechnology, Uppsala, Sweden) with molecular weights ranging from 14 400 to 94 000 was used as a standard.

Testing of biological activity in DLE preparations In vivo and in vitro methods were used for standardization of biological activity in the DLE preparations. In vivo testing of DLE preparations was conducted on white conventional SPF mice of live weight 18-20 g, challenged by S. typhimurium 4/5 strains (Mikula and Pistl, 1989) following the intraperitoneal application of DLE. The lowest DLE dose inhibiting the penetration ofS. typhimurium into the liver and spleen as well as the colonization of intestine in mice ( P < 0.01 ) was designated as the minimal inhibition dose (MID) (Mikula and Pistl, 1989). In vitro testing of biological activity of DLE preparations was conducted by the LMI test (Wilson et al., 1982) and the activity was expressed in potency units (PU) (Mikula et al., 1990).

Stabilization of DLE preparation A 10% apyretic bovine albumin in the ratio of 1 : 1 (Mikula et al., 1989) was used for the stabilization of the DLE preparation. The stability of DLE biological activity was tested after 6 and 12 months of storage at room temperature ( + 2 3 ° C ) and in the refrigerator ( + 4 ° C ) .

Testing of DLE stability on mouse Salmonella infection model A dose of 0.5 ml of the DLE preparation (a 10-fold dilution equal to OD26o-- 1.5 ) was injected intraperitoneally to the mice ( 18-20 g live weight, common SPF white breed). On Day 4 after DLE administration, the mice (l0 in one group), were orally infected with a dose of 5.103 CFU S. typhimurium, and on Day 8 post-infection, all mice were killed. The efficiency of DLE was evaluated in relation to the inhibition of penetration into the liver and spleen and colonization of the intestinal tract by the S. typhimurium 4/5 challenge strain. (Mikula and Pistl, 1989 ). Statistical evaluation was carded out using the Student's t-test.

Testing of DLE stability using the leukocyte migration-inhibition (LMI) assay Biological activity of the DLE was established by means of a modified capillary LMI assay according to the methods of Bendixen et al. (1976) and Wilson et al. (1982 ). Leukocytes from the blood of healthy calves were isolated by osmotic lysis of red blood cells (Karlson and Kaneko, 1973 ). Heat-inac-

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tivated S. typhimurium 4/5 strain in the broth culture (after three washings ) was used as an antigen at a concentration of 101° microorganisms m l Aliquots of 100/tl containing leukocytes at a concentration of 2.108 cells ml-~ were incubated for 60 min at 37 °C in Basal Eagle Medium (BEM) alone, in BEM with various DLE volumes (5-60 pl) and in BEM containing both heat-inactivated antigen and DLE (in different volumes). Based upon the titration, the antigen was added to leukocyte suspension at the ratio of 1 : 10. After 60 min of incubation, the 20/zl vol of individual leukocyte suspensions were aspirated into heparinized microhaematocrit capillaries. The capillaries were centrifuged ( 5 6 0 × g for 5 min), then cut at the interface of the sediment and supernatant. The capillary portion containing the cellular sediment was fixed by wax to the bottom of the wells of the plastic microplate (each well was 10 m m in diameter and 2 m m deep). The supernatant from the capillaries was blown out into wells that were filled to the upper margin with BEM containing 10% calf serum and antibiotics (penicillin 100 IU m l - 1 and streptomycin 100/tg m l - ~). These wells were then closed by a coverslide and incubated at 37°C for 18 h. The effect of antigen, DLE and the mixture of DLE plus antigen was quantitatively evaluated by determination of three migration indices termed, respectively, MI, MIA and MIB (Wilson et al., 1982 ). Responsiveness to antigen was expressed as a migration index (MI). The MIA value quantitatively determined the antigen-independent LMI. MIA values < 0.8 indicated the significant antigen-independent effects. The MIB value quantitated the antigen-dependent LMI induced by LIF (leukocyte-inhibition factor) released from T-lymphocytes newly sensitized by TF in the presence of specific antigen. An MIB value of < 0.90 indicated significant antigen-dependent LMI (Wilson et al., 1982). In this study all concentrations of DLE were evaluated in four replicate cultures• The MI, MIA and MIB values given below are the mean values __+standard deviation (SD) of all determinations. The antigen-dependent decrease of leukocyte migration was determined according to the formula: %Da = 1 - (MIB/MIA) × 100 (Wilson et al., 1982). The lowest volume of DLE that produced 20% antigen-dependent inhibition of the leukocyte migration was interpreted as one PU (Wilson et al., 1982; Fudenberg, 1987 ). The quantity of DLE potency units was determined as the number of this minimal DLE vol 1 m l RESULTS

The physical-chemical and biological characteristics of the DLE preparations are presented in Table 1. On the basis of the performed SDS-PAGE, we may assume that the tested DLE preparations did not contain proteins with mol wt > 10 0 0 0 (Fig. 1 ).

107

SALMONELLA-SPECIFICDIALYZABLELEUKOCYTEEXTRACTS TABLE 1 Physical-chemical and biological characteristics of the DLE preparations Variables

Preparations of DLE

Index OD26o/2s0 Ribose in 1 ml (Davidek et al., 1981) Proteins in 1 ml (Davidek et al., 1981) Content PU* in 1 ml per LMI assay (Wilson et al., 1982) MID**/mouse model (Mikula and Pistl, 1989)

DLE,-inf

DLES-im

DLE n

1.98 37.8/zg

2.2 43.3 #g

1.86 36.6/zg

3.9 nag 67 0.5 ml

3.8 mg 50 0.5 ml

4.0 mg 0 0

Characteristics: external form, white or light yellow powder; pH range, 5.85-6.60; microorganisms, sterile; pyrogenicity, negative. ~DLE'-t"f, Salmonella-specific bovine dialyzable leukocyte extract (DLE) prepared from mesenteric lymph nodes and spleens of calves immunized with Salmonella-vaccine and subsequently infected with S. typhimurium 4/5. DLE"-ira, Salmonella-specific bovine DLE prepared from fattening bulls immunized with Salmonella-vaccine. DLE", Non-specific DLE prepared from fattening bulls. PU*, Potency units in 1 mi of DLE, established in LMI assay according to formula: %DB= 1 - (MIa/ MIA) × 100 (Wilson et al., 1982). MI^, migration of leukocytes with DLE/migration ieukocytes without; MIB, migration of leukocytes with antigen + DLE/migration of leukocytes with DLE. Vol. of the DLE preparation with DLE produces 20% inhibition of LMI in an Ag-specific manner and determines the 1 PU. Used antigen: Salmonella typhimurium, heat-inactivated. MID**, Minimal inhibition dose. The lowest DLE dose inhibiting the penetration of Salmonella typhimurium into the liver and spleen as well as the colonization of intestine in mice (P< 0.01; Mikula and Pistl, 1989).

1

2

3

S

LMW (Pharmacia) 9 0 . 0 0 0 phosphorylase b 6 7 . 0 0 0 albumin 43. 000 ovoalbumJn 30.000 carbonic anhydrase 20.000 trypsin inhibitor 14.400 alfa lactalbumin

Fig. 1. S D S / P A G E of three DLE preparations according to Laemmli (1970). legend: 1. DLE'-I"f; 2. DLE s-ira, 3. DLE"; S, low molecular weight ( L M W ) calibration kit (Pharmacia).

1. M1KULA ET AL.

108 TABLE 2 Testing of Salmonella-specific bovine DLE effect on mouse model Storage period of DLE (in months )

0

6

Type of DLE tested

DLE DLE+albumin Albumin No treatment DLE+albumin DLE

12

No treatment DLE+albumin DLE

No treatment

Temperatures for storage (°C)

-20 + 4 + 4 +23 + 4 +23 + 4 -20 +23 + 4 +23 + 4 -20 -

S. typhimurium in 1 g of organ on Day 8 post-infection Liver

Spleen

Intestine

x_+SD

P<

~¢_+SD

P<

;?_+SD

P<

0.84__+ 1.02 1.04_+ 1.12 3.06+ 1.70 3.67_+ 1.77 3.25_+ 1.23 1.82_+0.81 2.78_+ 1.36 2.41 _+0.94 1.24+ 1.06 4.06-+ 1.44 2.96_+ 1.6 1.15_+1.1 3.34_+ 1.97 3.11_+1.9 0.93_+1.28 3.43_+ 1.68

0.001 0.01 0 0 0.01 0 0.05 0.001 0 0.01 0 0 0.01 -

1.20+ 1.08 0.87+0.96 2.78-+ 1.48 3.18_+ 1.6 3.12_+ 1.2 1.35_+0.9 2.8 _+ 1.12 2.72_+0.87 0.92-+ 1.26 3.58-+ 1.38 2.65_+ 1.46 0.98_+0.97 3.08_+ 1.98 2.65_+1.70 1.14_+1.30 2.96_+ 1.76

0.01 0.001 0 0 0.01 0 0 0.01 0 0.01 0 0 0.01 -

1.06+ 1.12 1.62-+0.93 3.89-+ 1.56 4.12_+ 1.51 3.86_+ 1.12 1.64_+1.20 4.3 _+0.94 4.08_+0.6 1.48-+1.53 4.51 _+ 1.75 3.88_+ 1.29 2.08_+1.03 3.93_+ 1.8 3.42_+1.65 1.18_+1.53 3.86_+ 1.38

0.001 0.01 0 0 0.01 0 0 0.01 0 0.05 0 0 0.01 -

DLE was applied at a dose of 0.5 ml i.p. at 4 days before oral infection with S. typhimurium 4/5 at a dose of 103 CFU 0.1 m l - ~. The statistical evaluation was performed by Student's t-test; P = level of statistical significance. DLE, dialyzable leukocyte extract.

TABLE 3 The determination of potency units (PU) in Salmonella-specific bovine DLE by means of capillary LMI assay Dose of DLE s-inf in/~1

% antigen-dependent inhibition of leukocyte migration (% Dn ) DLE storage with albumin

DLE storage without albumin -20°C

+4°C

+23°C

+4°C

+23°C

6mo

12mo

0mo

6mo

12mo

6mo

12mo

0mo

6too

12mo

6mo

12mo

10 15 20 25 30 35 40 45 50 55 60

18 18 22 24 24 24 24 23 26 27 23

18 12 10 18 22 21 23 22 24 27 24

18 23 23 27 26 23 22 26 29 23 24

0 3 0 5 0 2 6 2 4 9 8

0 4 3 0 0 0 0 0 4 0 11

0 0 9,5 0 3 1 7 4 3 4 2

0 0 8 2 0 2 0 2 0 0 1

16 15 24 24 24 22 24 21 20 21 23

5 1 5 4 8 12 19 22 23 28 24

4 7 5 6 7 9 13 18 23 22 21

0 5 0 0 0 2 0 2 5 0 0

4 5 7 6 0 4 0 6 3 7 3

PU m l - 1

50

33

66

0

0

0

0

50

22

20

0

0

PU, the potency units of I ml of DLE preparation based upon % DB. The values in the table were calculated according to formula: %Da= l - (MIa/MIA) × 100 (Wilson et al., 1982). MI A, migration of leukocytes with DLE/migration leukocytes without DLE; MIa, migration of leukocytes with antigen + DLE/migration ofleukocytes with DLE. The lowest vol. of DLE preparation which produces 20% inhibition of LMI in an antigen-specific manner determines the 1 PU. mo, months.

SALMONELLA-SPECIFICDIALYZABLELEUKOCYTE EXTRACTS

109

Salmonella-specific DLE kept at - 2 0 °C and DLE-stabilized by albumin after being stored at + 4 ° C for a period of 6 and 12 months induced a highly significant inhibition of Salmonella penetration into the liver and spleen and also inhibited the intestinal tract colonization of the mice. The DLE with albumin kept at + 23 °C and DLE without albumin stored at + 4 °C and 23 °C did not inhibit penetration of Salmonella into the liver and spleen and the same the colonization of intestinal tract (Table 2). The storage of DLE at - 2 0 °C and DLE stabilized with albumin at + 4 °C for 6 and 12 months enabled it to retain the required inhibition of leukocyte migration. The loss of DLE activity after 12 months of storage at - 20 ° C was less than 50% and in the case of DLE stabilized with albumin and stored at + 4 ° C represented a value of 60%. DLE without albumin, maintained at both temperatures (4°C and 23°C), as well as DLE stabilized with albumin and stored at + 23 °C, did not cause an inhibition of leukocyte migration in the presence of Salmonella antigen in the capillary LMI assay (Table 3 ). DISCUSSION

Immune system mediators include a majority of low-molecular DLE components (representing approximately 200 different substances of molecular weight < 10 000) comprising also cytokines. Dwyer ( 1987 ) isolated DLE from leukocytes obtained from individuals stimulated by antigen. Preparations isolated by him were capable of inducing a secondary immune response. Fudenberg ( 1987 ) described the DLE component of molecular weight 3500 as the antigen-specific transfer factor. Borkowsky et al. (1983) reported on induction-suppression DLE substances. Some DLE activities are presumably mediated by leukocytes produced by cytokines. Baker et al. (1990) described the recombinant bovine and porcine cytokines. Blecha (1990) predicted the use of cytokines in respiratory diseases and mastitis and recommended cytokines in prophylaxis or therapy for farm animals. DLE used in our studies represents a mixture of low-molecular substances isolated from leukocytes, obtained from individuals immunized with S. typhimurium vaccine and subsequently infected with S. typhymurium. This mixture displayed biological activity which we attempted to stabilize using albumin. Our future studies will be directed at a more detailed characterization of DLE composition, as well as the biological activity of its individual components. Our work presents a method of stabilization of DLE by bovine apyretic albumin, which enables the preservation of biological activity of DLE, provided that it is stored at 4°C. DLE is temperature-sensitive and the preservation of its biological activity requires storage temperatures from - 20 ° C to 70°C (Fudenberg, 1987 ), and this work therefore represents a considera-

110

1.MIKULAETAL.

ble advantage from the viewpoint o f storage and distribution requirements o f DLE. Bovine albumin has proved to be the stabilizing factor for DLE activity during the 12-month storage at + 4 ° C. Albumin is probably the best-known binder o f biological fluids (Peters, 1975 ). This protein binds, generally with low affinity, endogenous and exogenous molecules like thyroid or steroid hormones, bilirubin, free fatty acids, metals and xenobiotics such as drugs (Vallner, 1977; Kragh-Hannsen, 1981 ). Some proteins in plasma can be considered as specialized carriers for certain compounds. There is a very wide variety o f affinity between ligands and binding proteins (Alv~in, 1986). The pharmacological or physiological activity is generally attributed to the u n b o u n d moiety o f the drug or substance, while the b o u n d moiety represents a temporarily inactive store o f the c o m p o u n d or metal ion. The b o u n d substance is also protected from elimination (Alv~ln, 1986). Our results indicate that DLE stabilized with apyretic bovine albumin can retain its biological activity in storage at 4 ° C for a period o f 12 months.

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Davidek, J. Hrdli~a, J., Karv~tnek, M., Pokorn~, J., Seifert, J. and Veli~ek, J., 1981. Laborat6rni PHru~ka Anal~,zy Potravin. SNTL, Prague, pp. 182-183,250-25 I. Dwyer, J.M., 1987. Transfer factor modifies cell mediated immunity by an anti-idio typic mechanism: a hypothesis for antigen specificity. In: V. Mayer and J. Borv~ik (Editors), Proceedings of the Fifth International Symposium on Transfer Factor, 10-13 November 1986, Smolenice, Slovak Academy of Sciences, Bratislava, pp. 324-332. Fudenberg, H.H., 1987. Transfer factor: Past, present and future. Opening lecture. In: V. Mayer and J. Borv~tk (Editors), Proceedings of the Fifth International Symposium of Transfer Factor. Smolenice, 10-13 November 1986, Slovak Academy of Sciences, Bratislava, Suppl., pp. XX-IL. Huo, Bao-Lai, Liu, Xiao-Fen, Wang, Hui-Fong, Liu, Zhao-Xian and Zhang, Xue-Kven, 1987. Laboratory analysis of transfer factor prepared from bovine spleens. In: V. Mayer and J. Borv~ik (Editors), Proceedings of the Fifth International Symposium on Transfer Factor. Smolenice, 10-13 November 1986, Slovak Academy of Sciences, Bratislava, pp. 31-44. Karlson, G.P. and Kaneko, J.P., 1973. Isolation of leucocyte from bovine peripheral blood. Proc. Soc. Exp. Biol. Med., 142: 853-856. Klesius, P.H., 1987. Bovine transfer factor use in protozoan diseases. In: V. Mayer and J. Borv~lk (Editors), Proceedings Fifth International Symposium on Transfer Factor. Smolenice, 10-13 November 1986, Slovak Academy of Sciences, Bratislava, pp. 203-224. Kragh-Hansen, U., 1981. Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev., 1: 17-53. Laemli, V.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T h. Nature, 227: 680-685. Lawrence, H.S., 1955. The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with disrupted leukocytes. J. Clin. Invest., 34:219. Mayer, V., Gajdo~ov~, E., Valaskov~i, M. and Oravec, C., 1985. Antigen-specific transfer factor from mice immunized with attenuated Flavivirus: augmentation of inducing activity in semipurified splenocytic dialyzates. Acta Virol., 29: 25-34. Mikula, I., Rosocha, J., Bulik, J. and Bre[tansk~, I., 1989. Intravenous preparation on the basis of albumin designated "Albulyzin i.v." during the treatment of diarrhoeic syndrome of calves. Folia Vet., 33(2): 59-70. Mikula, I. and Pistl, J., 1989. The use of mouse model for testing of Salmonella-specific leucocyte dialyzate. Acta Vet. Brno, 58:281-296. Mikula, I., Pistl, J. and Rosocha, J., 1992. Dialyzable leucocyte extract used in the prevention of Salmonella infection in calves. Vet. Immunol., 32:113-124. Mobarhan, S., 1988. The role of albumin in nutritional support. J. Am. Cell. Nutr., 7: 445-452. Pek~trek, J. and (~ech, K., 1986. Transfer factor SEVAC - possibilities and perspectives of its therapeutical using. Zpr~vy SEVAC, 1:3-34 (in Czech). Peters, T., 1975. Serum albumin. In: F.W. Putnam (Editor), The Plasma Proteins. Vol. 1, 2rid edn. Academic Press, New York, pp. 133-181. Remy, M.H. and Piznansky, M.J., 1978. Immunogenicity and antigenicity ofsolubile polymers: advantages for enzyme therapy. Lancet, 8: 68-70. Vallner, J.J., 1977. Binding of drugs by albumin and plasma protein. J. Pharmacol. Sci., 66: 447-466. Wilson, B.G., Welch, T.M. and Fudenberg, H.H., 1976. Human transfer factor in guinea pigs: partial purification of the active component. In: M.S. Ascher, A.A. Gottlieb and C.H. Kirkpatrick (Editors), Transfer factor: Basic Properties and Clinical Applications. Academic Press, New York. Wilson, G.B., Metcalf, H.F. and Fudenberg, H.H., 1982. Treatment of Mycobacterium fortuiturn pulmonary infection with "Transfer Factor" (TF): new methodology for evaluating TF potency and predicting clinical response. Clin. Immunol. Immunopathol., 23:478-491.

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Wilson, G.B., Newel, R.T. and Burdash, N.M., 1983. Immunochemical and physical-chemical evidence for the presence of thymosin alpha 1 peptide in dialyzable leukocyte extracts. In: C.H. Kirkpatrick, H.S. Lawrence and D.R. Burger (Editors), Fourth International Transfer Factor Workshop in Denver. Academic Press, New York. Wilson, G.B. and Fort, J.B., 1987. Interspecies transfers of cell-mediated immunity using specific immunity inducers with known potency-prevention and therapy in selected diseases. In: V. Mayer and J. Borv~ik (Editors), Proceedings of the Fifth International Symposium of Transfer Factor. Smolenice, 10-13 November 1986, Slovak Academy of Sciences, Bratislava, pp. 333-358. Zailian, L., 1987. Studies on porcine spleen cell dialyzates. In: V. Mayer and J. Borv~ik (Editors), Proceedings of the Fifth International Symposium on Transfer Factor. Smolenice, 1013 November 1986, Siovak Academy of Sciences, Bratislava, pp. 84-97.