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Studies on the Chemical Nature of Dialysable Transfer Factor. Comparison of Human Leukocyte Dialysate and Dialysates Derived from Human Serum and from Mammalian Lymphoid and Non-Lymphoid Organs
Immunobiol., vol. 156, pp. 353-363 (1979) Institute of Biomedical Biomedical Sciences, University of Tampere, Finland
Studies on the Chemical Nature of Dialysable Transfer Factor. Comparison of Human Leukocyte Dialysate and Dialysates Derived from Human Serum and from Mammalian Lymphoid and Non-Lymphoid Organs ARJA UOTILA
Received October 6, 1978 . Accepted July 26, 1979
Abstract The chemical nature of human dialysable transfer factor factor (TF d), d) , capable of augmenting mammalian organ organ dialysates, delayed hypersensitivity (DH) in human recipients, and some mammalian known to augment DH in antigen-primed guinea pigs, were compared using chromatography on Sephadex G-10 G-IO and G-25 columns and on thin-layer plates plates.. The fractions of human leukocyte dialysate which eluted eluted at or or after the Vt of the Sephadex columns have previously been shown to contain the in vivo TFd-activity and therefore special attention was paid to corresponding dialysate fractions. All together 52 identified or unidentified components were found found at or close to this elution region with thin-layer chromatography (TLC). (TLC). The 14 14 identified substances substances were nucleobases, nucleobases , nucleosides, nucleosides, sugars and aromatic aromatic or heterocyclic amino acids. Unidentified components had similar staining staining characteristics as the identified identified ones on TLC. No evidence was found for the presence of peptides or nucleotides. There There were no components specific for human leukocyte dialysate. Several of the identified identified and unidentified humal dialysable leukocyte extract extract were common to all or nearly nearly all substances in fractions of humal substances dialysates.. The possibility that that some of the unidentified components might might be responsible for dialysates the in vivo effect of human leukocyte dialysate in man or guinea pig is discussed.
Introduction
Human dialyzable transfer factor (TF d) was originally described by H.S. who reported that leukocyte dialysates derived from individuals who in skin testings showed a positive delayed hypersensitivity reaction als to certain recall antigens could induce this reactivity in recipients previously shown to have a negative skin reaction (1). These observations suggested a reactivity. Subsequent studies, transfer of immunological specific antigen reactivity. indicated that in addition to this postulated, immunologically however, indicated specific action, TF d also augments pre-existing immune reactivities of the recipients.. It has been suggested that this non-specific effect may be recipients responsible for the clinical benefits of human leukocyte dialysate in certain immune deficiencies (2, 3, 4, 5). The chemical nature of the components responsible for both the immunologically specific transfer and augmenting activity and their relationship to the therapeutic effect of human leukocyte dialysate are not known. Several fractionation methods have been used to LAWRENCE,
354 . AR]A ARJA UOTILA
separate the active component(s) from the crude dialysate preparations, in particular chromotography on Sephadex G-I0 and Sephadex G-25 gels (6, 7,8,9,10,11,12,13). In such studies both the immunologically specific and nonspecific in vivo TF d activities were frequently located in fractions eluting after the total volume (Vt ) of the column (6, 7, 8, 9, 10, 11, 12, 13). On Sephadex G-I0 chromatography, TFd activity capable of augmenting skin reactivity in human recipients was found in fractions containing uracil (13), and in comparison on Sephadex G-25 chomatography, the activity was found in a hypoxanthine containing fraction (8, 9, 11). Human crude dialysable leukocyte extracts and their hypoxanthine containing fractions augmented also the tuberculin and SK-SD skin reactivity of guinea pigs primed with these antigens (14, 15, 11). In addition, our recent studies with primed guinea pigs show that mammalian lymphoid (porcine spleen) and non-lymphoid (bovine liver) dialysates augment the skin reactivity of antigen primed guinea pigs in a similar fashion as human dialysable leukocyte extract (16). It was therefore felt that a comparative chemical analysis of human leukocyte and various mammalian organ dialysates might reveal components responsible for the immunologically nonspecific augmenting activity of human dialysable leukocyte extract. As in previous studies by ourselves (13) and by others, gel filtration on Sephadex G-I0 and Sephadex G-25 and TLC have been used to compare different preparations of human leukocyte dialysate, some mammalian organ dialysate and human serum dialysate. Material and Methods Preparation of dielysstes dialysates Preparation from the Finnish Red Cross Cross Blood Human buffy coats (two preparations) were obtained from Transfusion Service and heparinised porcine blood (two preparations) from a slaughterhouse. After sedimentation of the buffy coats and porcine blood with an equal volume volume of 6 % dextran After (Macrodex, Leiras Pharmaceutical, Turku, Finland) at 37°C, the leukocyte-rich supernatants collected and the leukocytes were washed with Hanks' Balanced Salt Solution (Flow (Flow were collected added to ten buffy coats. The Laboratories, Scotland, U.K.). One ml of distilled water was added cells were distrupted by freezing and thawing ten times (±20 °C). ovary and prostate (one preparation of each) were obtained at autopsy within 4 Human ovary hours after the death. Porcine thymus, spleen, lymphnode, kidney and liver (two preparations of each) and bovine bovine spleen, thymus, lymphnode and liver (two preparations of each) were obtained from freshly killed animals from a slaughterhouse. Fat and connective tissue were trimmed from the organs, which which were minced minced with a scalpel and homogenized in cold distilled (Braun, Melsungen, Melsungen, West Germany). water in an Ultra Turrax homogenizer (Braun, Human AB+ serum (two preparations) was obtained from Finnish Red Cross Blood Transfusion Service. organ homogenates, and serum serum were centrifuged for 60 minutes at The leukocyte lysates, organ 100.000 g. The cleared supernatants were ultrafiltered at 4 °C through Visking dialysis tubing (American Carbide Co). The dialysates were lyophilized, weighed and stored at 4 °C in in a desiccator.
Gel filtration cm) and G-25 (column 2.5XI00 em) cm) (Pharmacia (Pharmacia Fine Sephadex G-I0 (column 1.6X90 ern) Chemicals, Uppsala, Sweden) were used with distilled water as an eluent at constant flow rate. Chemicals,
Chemical Characterization of Transfer Factor . 355
(Vo) Fine Chmicals) was 45 ml The outer volume (V o ) as estimated with Blue Dextran (Pharmacia Fine and 205 ml for G-25 G-25 columns. Kav-values Kav-values for different fraction were calculated for G-10 gel and from Ve-Vo Kav = V-V' , 0 in which V. Ve is the elution volume of the fraction. The total volume of the Sephadex G-10 G-10 181 ml, and that of the Sephadex G-25 G-25 column was 490 ml. ml. A 200 mgcolumn (V,=3tr2h) (Vt=l't~h) was 181 sample of each dialysate was fractioned on sample on a Sephadex G-10 G-10 column and a total volume of 360 ml of eluent was collected in 3-ml portions. In addition, a 500 mg-sample of human leukocyte dialysate was fractionated on on a Sephadex G-25 column, and and a total volume of 1000 ml of eluent was collected in 6-ml 6-ml portions. These were pooled according to peak pattern recorded at 280 nm, lyophilized and stored in a desiccator at +4 "C. dc. The areas of the peaks peaks in the nm and 260 nrn, -curves of the pooled Sephadex G-l0 A2f1 G-10 fractions were calculated by mulciplying multiplying 2f1r 28oO-curves r and A 2B by the width of the fraction in ml. the height of the peak in absorbance units units by m!.
TLC TLC was performed on precoated, fluorescent 20X20 em plates plates (Kieselgel F254, Merck, Darmstadt, West Germany). Plates were developed in chloroform-methanol-17% aqueous ammonium hydroxide (40 :40 :20), or in N-butanol-acetone-acetic Nsbutanol-acerone-acetic acid-5% acid-SOlo aqueous ammonium hydroxide-distilled water (35 :25: 15: 15: 10) (17). After the run the plates were UV light at 254 nm (Uvis, (Uvis, Desaga, West Germany). Spots absorbing at viewed in UV Desaga, Heidelberg, West 254 nm, or emitting fluorescent light at 366 nm, were recorded by photography, and the plates then stained with ninhydrin (Merck, Darmstadt, West then West Germany) ' or orcinol (17). The following reference compounds (Merck, Darmstadt, West Germany, pro analyse) analyse) were used of spots in both solvent systems: 1) noncyclic mononucleotides as free acids for identification of and sodium salts, 2) ribosyl and deoxyribosyl nucleosides, 3) pyrimidine and purine nucleobases and hypoxanthine, xanthine, uric acids, niacin niacin and nicotinamide, 4) D-ribose, DDdeoxyribose, D-saccharose, D-glucose, Dvfructose, D-fructose, D-maltose, L-ascorbic acid and 5) natural were made in distilled water. Both the L-amino acids. Solutions containing 1 mg or 0.2 mg/ml were identified spots and reference spots as well as most of the unidentified spots could be classified according to their staining behaviour into the following groups: A) orcinol positive and absorbing 254 nm light with or without emitting at 366 nm light (corresponding to nucleotides negative, but absorbing 254 nm light with or without emitting at and nucleosides), B) orcinol negative, 366 nm light (corresponding to heterocyclic bases), C) orcinol positive only (corresponding to simple sugars and in addition to orcinol positive substances with unknown structures) and D) ninhydrin positive only (corresponding to natural amino acids). In addition a few unidentified compounds could be E) classified classified as emitting at 366 nm light light only.
Hydrolysis Acid hydrolysis was performed by mixing mixing 200 !lg G-25 fractions 5--6 5-{', Acid ~g of each of Sephadex G-25 G-10 fractions VIa, VIb and VII VII of human leukocyte dialysate with 0.5 ml and 7 and Sephadex G-10 of 6.0 N HC!. HCl. The mixtures were placed screw cupped teflon tubes, covered with placed in 2 ml screw paraffin oil and sealed. After 100 hours at 110°C the hydrolysate was dried by by vacuum evaporation, solved solved in distilled water and analyzed on on TLC.
Results
filtration Gel Iiltruion Dialysates of leukocytes or of different organs yielded nine main peaks after chromatography on Sephadex G-10 and accordingly the chromato1). Two graphic run was divided into nine fractions, numbered I-IX (Fig. 1). peaks were seen in nine of 24 dialysates in fraction II, which was accord-
ARJA UOTILA 356 . ARjA
A 5.0
4.0
- - - - A 280 - - A 260
JZI. JZI. 3.0
2.0 2.0
1.0
100
200
300
ELUTION VOLUME (ML) (ML) ELUTION cm column of Figure 1. Fractionation of 200 mg of porcine Iymphnode dialysate on on 1.6 X 90 ern were I 48-72 ml, Sephadex G-I0 with distilled water as eluent. Elution volumes of the fractions were II 73-84 ml, III 85-99 ml, mI, IV 100-114 ml, V 115-150 ml, VIa 151-183 151-183 ml, mI, VIb 184-201 ml, mI, VII 202-261 ml, VIII 262-300 ml, IX 301-360 ml.
ratios of absorbance at Table 1. The means and standard deviations of the K,v-values and ratios Each mean has been 260 nm and 280 nm for Sephadex G-I0 gel filtration fractions I-IX. Each calculated from the gel filtration results of 24 dialysates from various sources (without AB Rh + plasma) Fraction number
K,v mean ± S.D.
I IIa lIb') III III IV V VIa VIbb) VIle) VIII IX
0.09 0.21 0.25 0.37 0.48 0.63 0.81 1.01 1.15 1.70 2.02
± ± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.03 0.04 0.05 0.06 0.10 0.15 0.09 0.10 0.23
260 nm/280 mean ± S.D. 1.06 1.24 1.23 1.79 1.72 1.11 3.43 2.61 7.21 7.21 1.30 0.66
± ± ± ± ± ± ± ± ± ± ±
0.12 0.20 0.15 1.46 0.64 0.70 0.95 1.54 2.26 0.37 0.24
') Human ovary, porcine thymus and liver and bovine spleen spleen and lyrnphnode Iymphnode dialysates b) Porcine leukocyte and porcine thymus dialysates contain no fraction contain no fraction lIb. b) e) Bovine Bovine spleen and lymphnode dialysates contain no fraction VII. VIb. e)
Chemical Characterization of Transfer Factor . 357
ingly divided in two subfractions, IIa and lIb. Fraction VI was similarly divided to VIa and VIb in twenty of 24 dialysates. With human serum dialysate, no absorption at 260 or 280 nm was seen in the elution area corresponding to fractions VIa-IX. The means of the Kav-values and the rations of absorbances at 260 and 280 nm of each Sephadex G-l0 fractions are shown in table 1. The areas of each fraction varied greatly between individual dialysates and thus the standard deviation of the means were greater than 100 per cent for most of the fractions. Variation was also seen in the ratio of absorbances at 2601280 nm indicating that the fractions differed not only quantitatively, but also qualitatively from each others. The fractionation of human leukocyte dialysate on Sephadex G-25 gel yielded seven main peaks numbered 1-8, similar to an earlier report (13).
TLC of Sephadex fractions A detailed analysis of TLC was only performed for fractions IV-IX of Sephadex G-l0 and for fractions 4-8 of Sephadex G-25. A total of 52 different TLC-spots were seen in the Sephadex G-l0 fractions; 8 were found in fraction IV, 9 in fraction V, 3 in fraction VIa, 12 in fraction VIb, 5 in fraction VII, 6 in fraction VIII and 9 in fraction IX. 14 of the TLC-spots could be tentatively identified with the aid of the reference substances used. The reference nucleotides had Rf-values ranging from < 0.1 0.1 to 0.04, and no spots with these Rf-values were seen in fractions IV-IX. A schemativ TLCpicture of the compounds in fractions VIa, VIb and VII is shown in figure 2. F rae t ion Fraction ViaVI a 1----
Rf 1.0
0.9
'
0.9 2
0.7
•.
Rf Rt
Fraction Vlb
1.0
Nicolln-
•
:lp
3'-riacin
0.6 0.5 0.4
.a 0.2
F rae ti 0 VII n VII Fraction
i.
5_
12. n
AD
0 .a
0.2
Glucose
0.1.
0.1
Orcinol pOSitive
t.
Figure 2. Schematic thin-layer chromatograms of fractions VIa, VIb and VII from Sephadex G-IO chromatography. Each substance is located in the fraction where the strongest spot was obtained with orcinol staining has been marked as follows: seen most frequently. The color obtained = turquous, specific for ribose and ribosyl nucleosides or nucleotides, ,r((11;-; ,r;:?l!'; = all non.":,~!,, specific colors (brown, green, bright bright yellow, purple). .,,,,~!," specific
e
Rf Rt 1.0
5-(, 5--6 5-(, 5-6 not found found 7 not found 7 7 not found not 5-6 5--6 7 not found not not found not found not found not found not not found found not found not found not found found 7 not not found
VIal VIa2 VIa2 VIa, VIa3 VIb l vrs, Vlb22 VIb vn, VIb, Vlb4 vrs, Vlbs vu, Vlb6 VIb Vlb7 vis, vis, VIbs vn, VIb9 VIb lo vrs., VIb ll VIb Vl b 12 VIII VII 22 VII VII,3 VII 4 VIIs
Hypoxanthine
Glucose
Niacin
Nicotinamide
Thymine Uracil Uracil
Compon ent identical with reference substance substancess
C B A B A C C C E B B B A
B B A B B B C
Staining behavioural r')
+ + +
+ +
+ ++
+
+
+ +
+ +
+
+ +
Leukocytes Human Porcine Porcine with TF TF-activityb) activity")
+
+
Serum Human
+
+
+ +
+ + + + +
+
+
+
+ + +
+
+ + + + +
+ +
Lympho id tissues Porcine Bovine
+ +
+ +
+
+ + + + +
+
+
Ovine
Sources of dialysates
+ +
+ +
+ + +
+ + + +
+
+ + +
+ + + + + + + +
+
+ +
+
+
+ + +
Non-lym phoid tissues Human Porc Porcine ine Bovine
.) A) orcinol positive a) positive and absorbing 254 nm light with with or without emitting emitting at 366 nm light, B) orcinol orcinol negative, but but absorbing 254 nm light with or without emitting at 366 nm light, C) orcino orcinoll positive only, only, D) ninhydrin positive, E) at 366 nm light emittance emittanc e only. b) Open spaces = no detectable spots.
Localization in G-25 fractions fract ions
Spot Spot number in G-IO fractions fractions
Table 2. Occurre nce of the components in Sephadex G-10 Table G-IO fractions VIa, VIb, Vlb, and VII of and Sephadex G-25 G-2S fractions 5--6 S-f> and in 7 dialysates from leukocyt e, serum, lymphoi d and non serum, non-lym -lympho phoid id organs
:> >
f;:~
§::l0
C c::
:>-
Z! ~
00 00
~
...'-"en
not found not found not found 4 4 not found 4 s-6 5-6 4 not found not not found not found 5~ 5-6 5-6 5-6 not found not not found not found not not found not not found not found not found not not found not not found 8 8 8 8 8 8 8 8 8
IV IV!1 IV 2 IV 33 IV44 IV IVss IV 66 IV 7 IVa IVs V! VI V2 V V33 V4 V Vss V V66 V7 Vs VB V V99 VIII! VIllI VIII 2 VIII)3 VIII VIII VIII44 VIlIs VIII VIII66 IX IX!1 IX IX2 IX IX33 IX44 IX IX IXss IX IX66 IX IX7 IX IXsB
a) See Table 2. .)
I~
Localization G-25 in G -25 fractions fract ions
Spot number in G-I G-IO O fractions
Tryptophan
Guanosine
Tyrosine Inosine
Ribose Ribose Thymidine
Uridine
Phenylalanine
Component identical identical with with reference substances E C B D A A A C A A D B D A B B A B A A C A A A A A A D D D D A
Staining Staining behaviour') behaviour")
leukocyte, serum, lymphoid and non-lymphoid organs organs
+ + + + + + + + +
+
+
+ + +
+ + + + + +
+
+ + + + + + + + + + +
+
+ + +
+
+ +
+ + +
+ + + ++
++ ++ +
+
+ + + + + + ++ +
+ ++ ++ ++
+ + + +
+
Lymphoid tissues Bovine Porcine Bovine
+ +
+
+
Serum Serum Human
+ + + + +
+
+ +
++ + + +
Leukocyt es Leukocytes Human Porcine TFwith TFactivityb) acriviry")
+
+ + + +
+ + +
+
+ + +
+ + + ++
+ + +
+
++
+ +
+
++
++ ++
++
+
+ + ++ + + + + + + ++
+ + + + +
+
+
+
+ + + + ++
+
+
+
+ +
+ + +
+ +
+ + + +
Non-lymphoid tissues Human Porcine Bovine
+
Ovine
Sources Sources of dialysates
'"3
.... V> '" -0 '"
n
..08...
'Tl '11
..
;:t' ...
'"
OJ :I :> II>
'"
...j ...>-1
0 ..., .....
:> :I
~ s: o·
N
f; fti ::1 :! .
n
.'"
n::r o:T "...'"
e!-
n' e,
n::r o:T
ARJA UOTILA 360 . ARJA
A comprehensive comparison of the substances found in various dialysates, after fractionation with Sephadex and TLC is given in tables 2 and 3. None of the fractions tested had components specific for human leukocyte dialysate. However, several components in VIa, VIb and VII were or nearly all (thymine, nicotinamide, niacin, common to all (uracil, VIb 4) or or organ dialysates. VIb 6, VIb 7, glucose, hypoxanthine) cell or Human serum dialysate contained only a few substances in area corresponding to fraction IV-IX. The strongest spot in TLC represented glucose and this material was found in several fractions. Phenylalanine and tyrosine were seen in fractions IV and V, respectively. An unidentified spot in fractions VIa, VIb and VII of human serum dialysate corresponds to VIb 4 (table 2).
Acid hydrolysis 5--6 or or 7 following acid No change was seen in fraction VIa, VIb, VII, 5-6 hydrolyses except the decomposition of uracil and hypoxanthine. No new ninhydrin positive spots were seen, suggesting the absence of detectable amounts of peptides in these fractions.
Discussion In this work we have compared the chemical composition of human d) and other mammalian organ dialysates in order to leukocyte dialysate (TF (TFd) characterize the component(s) responsible for their in vivo effect on deleayed hypersensitivity in man or in guinea pig. The rationale of this or approach is based on the assumption that biologic similarity (16) or differences of the various dialysate would also reflect in their chemical composition. This approach is limited, however, by the fact that all dialysates so far tested (human leukocyte, porcine spleen and bovine liver dialysates) were active in vivo and no proven inactive preparations were available. Human serum dialysate was therefore used as a putative inactive preparation. While it is established that serum is inactive in transferring cellular immunity (18), the effect of serum or specific cellular or serum dialysate in the guinea pig model has not been tested. In this study the chemical analysis was restricted to the Sephadex G-I0 and Sephadex G-25 fractions which eluted at or or after the Vt of the columns, since most investigators have located their in vivo TF d activity of human leukocyte dialysate in both human recipients and antigen primed guinea pig, in this elution region. In this region both gels operate mainly on the adsorption principle, and the order of elution of identified as well as unidentified components are the same in both gels (table 2, 3), and thus results obtained with sephadex G-I0 and G-25 are most probably comparable. The study yielded no firm evidence indicating which component(s) were responsible for the augmenting effect in human leukocyte dialysate or or
361 Chemical Characterization of Transfer Factor . 361
in the guinea pig model with organ dialysates. The uracil containing fraction VIa and the hypoxanthine containing fraction VII as well as fraction VIb, which eluted in between these two larger peaks, contained several unidentified substances common to all or or nearly all dialysates (table 2). In contrast, or in human plasma dialysate contained hardly any spots in these fractions or geOl~ral. This could indicate that some of the the fraction eluting after Vt in general, dialy~ate were responsible for the in vivo effect substances missing in serum dialysate of human leukocyte dialysate in man. None of the spots in fractions VIa, VIb, VII were specific for human leukocyte dialysate, or for the two active organ dialysates derived from porcine spleen and bovine liver. The nature of the unidentified substances in the human leukocyte and unclear. In contrast to the findings of others mammalian organ dialysates is unclear. or mono/oligonucleotides mon%ligonucleotides were not found in the fractions (19, 20), peptides or TFdd activity (VI-VII). However, the present results with the known in vivo TF TF d are in accordance with those of Wilson et al. (11), who found that the TFd activity eluted with hypoxanthine in Sephadex G-25 chromatography. The active fraction, TF x ' which could be further separated from hypoxanthine (11), contained six unidentified components showing similar staining behaviour on TLC as the unidentified components found in the present work in Sephadex G-25 fractions 5-6 and 7. The presence of nicotinamide or close to the TFd's in vivo (21) and inosine (22) in fractions eluting at or activity was also confirmed in the present work. cqaracters in TLC thus support Sephadex chromatography and staining characters the view that the unidentified substances in fractions VIa, VIb, and VII from Sephadex G-l0 and in fractions 5-6 and 7 from Sephadex G-25 were similar to the substances tentatively identified in the present work, as nucleobases, nucleosides and related heterocyclic compounds. In addition, the comparison between the organ dialysates revealed that there were no components specific for human leukocyte dialysate or specific for human leukocyte, porcine spleen and bovine liver dialysate, all of which were capable in augmenting the skin reactivity of the guinea pigs and most of the identified and unidentified substances in in vivo active fractions of human leukocyte dialysate were common to all or nearly all organ dialysates. Although such small molecular components as hypoxanthine and nicotinamide are known to effect lymphocyte functions in vivo (21,23), it dremains to be studied whether or not they are related to the in vivo TF dactivity.
Acknowledgements from Sigrid Juselius Juselius Foundation and Emil The work has been supported with grants from Aaltonen Foundation. The skilled assistance of Mr. PEKKA VIRTANEN, Mr. YOSHIO KAvANOVA, Mr. RALPH EUA KYROLA is gratefully acknowledged. ASHORN and Miss EI]A
ARJA VOTILA 362 . AR]A
References H.S. 1963. 1963 . Transfer of immunological information in humans with dialysates 1. LAWRENCE, H.S. of leucocyte extract. Trans. Assoc. Am. Physicians 76: 84-91. 2. LEVIN, A.S., L.E. SPITLER, D.P. STITES, and H.H. FUDENBERG. 1971. Molecular intervenInvest. 50: tion in genetically determined cellular immune deficiency disorders. J. Clin. Invest. 59a. 3. VALDIMARSSON, H., G. HAMBLETON, K. HENRY, and J. MCCONNELL. 1974. Restoration extract. Clin, Clin. expo expo Immunol. 16: of T-lymphocyte deficiency with dialysable leucocyte extract. 141-152. 4. ARALA-CHAVES, M.P., R. PROENCA, and M. DE SOUSA. 1974. Transfer factor therapy in a case of complex immunodeficiency. Cell. Immunol. 10: 371-389. Defects. 11: 5. GRISCELLI, C. 1975. Transfer factor therapy in immunodeficiency. Birth Defects. 462-464. P.E. HURTUBISE, S.G. MURPHY, E.N. METZ, S.P. 6. NEIDHART, J.A., R.S. SCHWARTZ, P.E. BALCERZAK, and A.F. LoBuGLIO. 1973. Transfer factor: Isolation of a biologically active component. Cell. Immunol. 9: 319-323. J.A. NEIDHART, S.P. BALCERZAK, and A.F. LoBuGLIO. 1974. 7. ZUCKERMAN, K.S., J.A. Immunologic specificity of transfer factor. factor. J. Clin. Invest. 54: 997-1000. 8. TOMAR, R.H., R. KNIGHT, and M. STERN. 1976. Transfer factor: Hypoxanthine is a major component of a fraction with in vivo activity. J. Allergy din. Immunol. 58: 190-197.
The identification and 9. KIRKPATRICK, C.H., L.B. ROBINSON, and T.K. T.K. SMITH. 1976. The factor. Cell. Immunol. 24: 230-240. significance of hypoxanthine in dialyzable transfer factor. W.A. ANDERSON Jr., Jr., R.M. VETTO, and P. 10. BURGER, D.R., A.A. VANDENBARK, D. DAVES, W.A. FINKE. 1976. Human transfer factor: fractionation and biologic activity. J. Immunol. 117: 789-823. 789-823. T.M. WELCH, D.R. KNAPP, A. HORSMANHEIMO, and H.H. FUDENBERG. 11. WILSON, G.B., T.M. factor. 1. 1977. Characterization of Tx, an active subfraction of human dialyzable transfer factor. Identification of the major component in TFg, a precursor of Tx, as hypoxanthine. Clin. Immunol. Immunopathol. 8: 551-568. 12. GOTTLIEB, A.A., L.G. FOSTER, and S.R. WALDMAN. 1973. What is transferfactor? Lancet: 822-823. GROHN. 1977. Studies on 13. KROHN, K., A. VOTILA, J. VAISANEN, and P. GROHN. on the chemical composition and biological properties of transfer factor. factor. Z Immun. Forsch.- Immunobiol. 153: 395-411. 14. WELCH, T.M., R. TRIGLIA, L.E. SPInER, and H.H. FUDENBERG. 1976. Preliminary Systemic transfer of cutaneous studies on on human "transfer factor" activity in guinea pigs. Systemic Clin. Immunol. Immunopathol. 5: delayed-type hypersensitivity to PPD and SK-SD. Clin, 407-415. factor activity in 15. VANDENBARK, A.A., D.R. BURGER, R.M. VETTO. 1977. Human transfer factor specificity. Clin. Immunol. Immunopathol. 8: 7-16. the guinea pig: absence of antigen specificity. KAIuruMAKI. 1979. Augmentation of skin 16. KROHN, K., A. VOTILA, R. ASHORN and E. KARHuMAKI. reactivities in antigen primed guinea guinea pigs by transfer factor factor and other cellular dialysates. dialysates. reactivities Khan, C. Kirkpatrick, N.O. Hill) Hill) Immune Regulators in Transfer Factor (eds. A. Khan, Press, 655-660. Academic Press, 17. STAHL, E. (ed.) 1969. Thin-layer chromatography. Springer-Verlag. M.W. CHASE. 1942. Experiments on on transfer of cutaneous sensitivsensitiv18. LANDSTEINER, K., and M.W. ity chemical compounds. Proc. Proc. Soc. expo expo BioI. N.Y. 49: 688~90. ity to some chemical KHAN, A., O. GARRISON, J.M. J.M. HILL, HILL, and R.W. GRACY. 1977. Isolation and purification of 19. KHAN,A., immunopeptide from from transfer factor. factor. Exp. Haem. 5: 92. M.M. MOSKO. 1966. Studies on the transfer factor factor of human 20. BARAM, P., L. YUAN, and M.M. 1. Partial purification and characterization of two active delayed-type hypersensitivity. 1. components. J. Immunol. 97: 407-420.
Chemical Characterization of Transfer Factor . 363 Jr., R.M. VETTO, and P. 21. BURGER, D.R., A.A. VANDENBARK, D. DAVES, W.A. ANDERSON Jr., 1976. Nicotinamide: Suppression of lymphocyte transformation with a component FINKE. 1976. identified in human transfer factor. J. Immunol. ImmunoI. 117: 797-801. and R.M. YETTO. 22. VANDENBARK, A.A., D.R. BURGER, D.L., DREYER G.D. DAVES Jr., and VETTO. and high pressure reverse 1977. Human transfer factor: fractionation by electrofocusing and phase chromatography. J. Immunol. ImmunoI. 118: 636-641. 636--641. 23. HOVI, K.O. RAIVIO, and A. VAHERl. YAHERl. Purine metabolism and and control of HOVI, T., A.C. ALLISON, K.O. cell proliferation. Purine and pyrimidine metabolism, pp. 225-248. Ciba Foundation Symposium 48. Elsevier. Amsterdam. 1977. Dr. AR]A UOTILA, Institute of Biomedical Sciences, University of Tampere, P.O. Box 607, SFDr. 33101 Tampere 10, Finland. 33101
Studies on the Chemical Nature of Dialysable Transfer Factor. Comparison of Human Leukocyte Dialysate and Dialysates Derived from Human Serum and from Mammalian Lymphoid and Non-Lymphoid Organs ARJA UOTILA
Received October 6, 1978 . Accepted July 26, 1979
Abstract The chemical nature of human dialysable transfer factor factor (TF d), d) , capable of augmenting mammalian organ organ dialysates, delayed hypersensitivity (DH) in human recipients, and some mammalian known to augment DH in antigen-primed guinea pigs, were compared using chromatography on Sephadex G-10 G-IO and G-25 columns and on thin-layer plates plates.. The fractions of human leukocyte dialysate which eluted eluted at or or after the Vt of the Sephadex columns have previously been shown to contain the in vivo TFd-activity and therefore special attention was paid to corresponding dialysate fractions. All together 52 identified or unidentified components were found found at or close to this elution region with thin-layer chromatography (TLC). (TLC). The 14 14 identified substances substances were nucleobases, nucleobases , nucleosides, nucleosides, sugars and aromatic aromatic or heterocyclic amino acids. Unidentified components had similar staining staining characteristics as the identified identified ones on TLC. No evidence was found for the presence of peptides or nucleotides. There There were no components specific for human leukocyte dialysate. Several of the identified identified and unidentified humal dialysable leukocyte extract extract were common to all or nearly nearly all substances in fractions of humal substances dialysates.. The possibility that that some of the unidentified components might might be responsible for dialysates the in vivo effect of human leukocyte dialysate in man or guinea pig is discussed.
Introduction
Human dialyzable transfer factor (TF d) was originally described by H.S. who reported that leukocyte dialysates derived from individuals who in skin testings showed a positive delayed hypersensitivity reaction als to certain recall antigens could induce this reactivity in recipients previously shown to have a negative skin reaction (1). These observations suggested a reactivity. Subsequent studies, transfer of immunological specific antigen reactivity. indicated that in addition to this postulated, immunologically however, indicated specific action, TF d also augments pre-existing immune reactivities of the recipients.. It has been suggested that this non-specific effect may be recipients responsible for the clinical benefits of human leukocyte dialysate in certain immune deficiencies (2, 3, 4, 5). The chemical nature of the components responsible for both the immunologically specific transfer and augmenting activity and their relationship to the therapeutic effect of human leukocyte dialysate are not known. Several fractionation methods have been used to LAWRENCE,
354 . AR]A ARJA UOTILA
separate the active component(s) from the crude dialysate preparations, in particular chromotography on Sephadex G-I0 and Sephadex G-25 gels (6, 7,8,9,10,11,12,13). In such studies both the immunologically specific and nonspecific in vivo TF d activities were frequently located in fractions eluting after the total volume (Vt ) of the column (6, 7, 8, 9, 10, 11, 12, 13). On Sephadex G-I0 chromatography, TFd activity capable of augmenting skin reactivity in human recipients was found in fractions containing uracil (13), and in comparison on Sephadex G-25 chomatography, the activity was found in a hypoxanthine containing fraction (8, 9, 11). Human crude dialysable leukocyte extracts and their hypoxanthine containing fractions augmented also the tuberculin and SK-SD skin reactivity of guinea pigs primed with these antigens (14, 15, 11). In addition, our recent studies with primed guinea pigs show that mammalian lymphoid (porcine spleen) and non-lymphoid (bovine liver) dialysates augment the skin reactivity of antigen primed guinea pigs in a similar fashion as human dialysable leukocyte extract (16). It was therefore felt that a comparative chemical analysis of human leukocyte and various mammalian organ dialysates might reveal components responsible for the immunologically nonspecific augmenting activity of human dialysable leukocyte extract. As in previous studies by ourselves (13) and by others, gel filtration on Sephadex G-I0 and Sephadex G-25 and TLC have been used to compare different preparations of human leukocyte dialysate, some mammalian organ dialysate and human serum dialysate. Material and Methods Preparation of dielysstes dialysates Preparation from the Finnish Red Cross Cross Blood Human buffy coats (two preparations) were obtained from Transfusion Service and heparinised porcine blood (two preparations) from a slaughterhouse. After sedimentation of the buffy coats and porcine blood with an equal volume volume of 6 % dextran After (Macrodex, Leiras Pharmaceutical, Turku, Finland) at 37°C, the leukocyte-rich supernatants collected and the leukocytes were washed with Hanks' Balanced Salt Solution (Flow (Flow were collected added to ten buffy coats. The Laboratories, Scotland, U.K.). One ml of distilled water was added cells were distrupted by freezing and thawing ten times (±20 °C). ovary and prostate (one preparation of each) were obtained at autopsy within 4 Human ovary hours after the death. Porcine thymus, spleen, lymphnode, kidney and liver (two preparations of each) and bovine bovine spleen, thymus, lymphnode and liver (two preparations of each) were obtained from freshly killed animals from a slaughterhouse. Fat and connective tissue were trimmed from the organs, which which were minced minced with a scalpel and homogenized in cold distilled (Braun, Melsungen, Melsungen, West Germany). water in an Ultra Turrax homogenizer (Braun, Human AB+ serum (two preparations) was obtained from Finnish Red Cross Blood Transfusion Service. organ homogenates, and serum serum were centrifuged for 60 minutes at The leukocyte lysates, organ 100.000 g. The cleared supernatants were ultrafiltered at 4 °C through Visking dialysis tubing (American Carbide Co). The dialysates were lyophilized, weighed and stored at 4 °C in in a desiccator.
Gel filtration cm) and G-25 (column 2.5XI00 em) cm) (Pharmacia (Pharmacia Fine Sephadex G-I0 (column 1.6X90 ern) Chemicals, Uppsala, Sweden) were used with distilled water as an eluent at constant flow rate. Chemicals,
Chemical Characterization of Transfer Factor . 355
(Vo) Fine Chmicals) was 45 ml The outer volume (V o ) as estimated with Blue Dextran (Pharmacia Fine and 205 ml for G-25 G-25 columns. Kav-values Kav-values for different fraction were calculated for G-10 gel and from Ve-Vo Kav = V-V' , 0 in which V. Ve is the elution volume of the fraction. The total volume of the Sephadex G-10 G-10 181 ml, and that of the Sephadex G-25 G-25 column was 490 ml. ml. A 200 mgcolumn (V,=3tr2h) (Vt=l't~h) was 181 sample of each dialysate was fractioned on sample on a Sephadex G-10 G-10 column and a total volume of 360 ml of eluent was collected in 3-ml portions. In addition, a 500 mg-sample of human leukocyte dialysate was fractionated on on a Sephadex G-25 column, and and a total volume of 1000 ml of eluent was collected in 6-ml 6-ml portions. These were pooled according to peak pattern recorded at 280 nm, lyophilized and stored in a desiccator at +4 "C. dc. The areas of the peaks peaks in the nm and 260 nrn, -curves of the pooled Sephadex G-l0 A2f1 G-10 fractions were calculated by mulciplying multiplying 2f1r 28oO-curves r and A 2B by the width of the fraction in ml. the height of the peak in absorbance units units by m!.
TLC TLC was performed on precoated, fluorescent 20X20 em plates plates (Kieselgel F254, Merck, Darmstadt, West Germany). Plates were developed in chloroform-methanol-17% aqueous ammonium hydroxide (40 :40 :20), or in N-butanol-acetone-acetic Nsbutanol-acerone-acetic acid-5% acid-SOlo aqueous ammonium hydroxide-distilled water (35 :25: 15: 15: 10) (17). After the run the plates were UV light at 254 nm (Uvis, (Uvis, Desaga, West Germany). Spots absorbing at viewed in UV Desaga, Heidelberg, West 254 nm, or emitting fluorescent light at 366 nm, were recorded by photography, and the plates then stained with ninhydrin (Merck, Darmstadt, West then West Germany) ' or orcinol (17). The following reference compounds (Merck, Darmstadt, West Germany, pro analyse) analyse) were used of spots in both solvent systems: 1) noncyclic mononucleotides as free acids for identification of and sodium salts, 2) ribosyl and deoxyribosyl nucleosides, 3) pyrimidine and purine nucleobases and hypoxanthine, xanthine, uric acids, niacin niacin and nicotinamide, 4) D-ribose, DDdeoxyribose, D-saccharose, D-glucose, Dvfructose, D-fructose, D-maltose, L-ascorbic acid and 5) natural were made in distilled water. Both the L-amino acids. Solutions containing 1 mg or 0.2 mg/ml were identified spots and reference spots as well as most of the unidentified spots could be classified according to their staining behaviour into the following groups: A) orcinol positive and absorbing 254 nm light with or without emitting at 366 nm light (corresponding to nucleotides negative, but absorbing 254 nm light with or without emitting at and nucleosides), B) orcinol negative, 366 nm light (corresponding to heterocyclic bases), C) orcinol positive only (corresponding to simple sugars and in addition to orcinol positive substances with unknown structures) and D) ninhydrin positive only (corresponding to natural amino acids). In addition a few unidentified compounds could be E) classified classified as emitting at 366 nm light light only.
Hydrolysis Acid hydrolysis was performed by mixing mixing 200 !lg G-25 fractions 5--6 5-{', Acid ~g of each of Sephadex G-25 G-10 fractions VIa, VIb and VII VII of human leukocyte dialysate with 0.5 ml and 7 and Sephadex G-10 of 6.0 N HC!. HCl. The mixtures were placed screw cupped teflon tubes, covered with placed in 2 ml screw paraffin oil and sealed. After 100 hours at 110°C the hydrolysate was dried by by vacuum evaporation, solved solved in distilled water and analyzed on on TLC.
Results
filtration Gel Iiltruion Dialysates of leukocytes or of different organs yielded nine main peaks after chromatography on Sephadex G-10 and accordingly the chromato1). Two graphic run was divided into nine fractions, numbered I-IX (Fig. 1). peaks were seen in nine of 24 dialysates in fraction II, which was accord-
ARJA UOTILA 356 . ARjA
A 5.0
4.0
- - - - A 280 - - A 260
JZI. JZI. 3.0
2.0 2.0
1.0
100
200
300
ELUTION VOLUME (ML) (ML) ELUTION cm column of Figure 1. Fractionation of 200 mg of porcine Iymphnode dialysate on on 1.6 X 90 ern were I 48-72 ml, Sephadex G-I0 with distilled water as eluent. Elution volumes of the fractions were II 73-84 ml, III 85-99 ml, mI, IV 100-114 ml, V 115-150 ml, VIa 151-183 151-183 ml, mI, VIb 184-201 ml, mI, VII 202-261 ml, VIII 262-300 ml, IX 301-360 ml.
ratios of absorbance at Table 1. The means and standard deviations of the K,v-values and ratios Each mean has been 260 nm and 280 nm for Sephadex G-I0 gel filtration fractions I-IX. Each calculated from the gel filtration results of 24 dialysates from various sources (without AB Rh + plasma) Fraction number
K,v mean ± S.D.
I IIa lIb') III III IV V VIa VIbb) VIle) VIII IX
0.09 0.21 0.25 0.37 0.48 0.63 0.81 1.01 1.15 1.70 2.02
± ± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.03 0.04 0.05 0.06 0.10 0.15 0.09 0.10 0.23
260 nm/280 mean ± S.D. 1.06 1.24 1.23 1.79 1.72 1.11 3.43 2.61 7.21 7.21 1.30 0.66
± ± ± ± ± ± ± ± ± ± ±
0.12 0.20 0.15 1.46 0.64 0.70 0.95 1.54 2.26 0.37 0.24
') Human ovary, porcine thymus and liver and bovine spleen spleen and lyrnphnode Iymphnode dialysates b) Porcine leukocyte and porcine thymus dialysates contain no fraction contain no fraction lIb. b) e) Bovine Bovine spleen and lymphnode dialysates contain no fraction VII. VIb. e)
Chemical Characterization of Transfer Factor . 357
ingly divided in two subfractions, IIa and lIb. Fraction VI was similarly divided to VIa and VIb in twenty of 24 dialysates. With human serum dialysate, no absorption at 260 or 280 nm was seen in the elution area corresponding to fractions VIa-IX. The means of the Kav-values and the rations of absorbances at 260 and 280 nm of each Sephadex G-l0 fractions are shown in table 1. The areas of each fraction varied greatly between individual dialysates and thus the standard deviation of the means were greater than 100 per cent for most of the fractions. Variation was also seen in the ratio of absorbances at 2601280 nm indicating that the fractions differed not only quantitatively, but also qualitatively from each others. The fractionation of human leukocyte dialysate on Sephadex G-25 gel yielded seven main peaks numbered 1-8, similar to an earlier report (13).
TLC of Sephadex fractions A detailed analysis of TLC was only performed for fractions IV-IX of Sephadex G-l0 and for fractions 4-8 of Sephadex G-25. A total of 52 different TLC-spots were seen in the Sephadex G-l0 fractions; 8 were found in fraction IV, 9 in fraction V, 3 in fraction VIa, 12 in fraction VIb, 5 in fraction VII, 6 in fraction VIII and 9 in fraction IX. 14 of the TLC-spots could be tentatively identified with the aid of the reference substances used. The reference nucleotides had Rf-values ranging from < 0.1 0.1 to 0.04, and no spots with these Rf-values were seen in fractions IV-IX. A schemativ TLCpicture of the compounds in fractions VIa, VIb and VII is shown in figure 2. F rae t ion Fraction ViaVI a 1----
Rf 1.0
0.9
'
0.9 2
0.7
•.
Rf Rt
Fraction Vlb
1.0
Nicolln-
•
:lp
3'-riacin
0.6 0.5 0.4
.a 0.2
F rae ti 0 VII n VII Fraction
i.
5_
12. n
AD
0 .a
0.2
Glucose
0.1.
0.1
Orcinol pOSitive
t.
Figure 2. Schematic thin-layer chromatograms of fractions VIa, VIb and VII from Sephadex G-IO chromatography. Each substance is located in the fraction where the strongest spot was obtained with orcinol staining has been marked as follows: seen most frequently. The color obtained = turquous, specific for ribose and ribosyl nucleosides or nucleotides, ,r((11;-; ,r;:?l!'; = all non.":,~!,, specific colors (brown, green, bright bright yellow, purple). .,,,,~!," specific
e
Rf Rt 1.0
5-(, 5--6 5-(, 5-6 not found found 7 not found 7 7 not found not 5-6 5--6 7 not found not not found not found not found not found not not found found not found not found not found found 7 not not found
VIal VIa2 VIa2 VIa, VIa3 VIb l vrs, Vlb22 VIb vn, VIb, Vlb4 vrs, Vlbs vu, Vlb6 VIb Vlb7 vis, vis, VIbs vn, VIb9 VIb lo vrs., VIb ll VIb Vl b 12 VIII VII 22 VII VII,3 VII 4 VIIs
Hypoxanthine
Glucose
Niacin
Nicotinamide
Thymine Uracil Uracil
Compon ent identical with reference substance substancess
C B A B A C C C E B B B A
B B A B B B C
Staining behavioural r')
+ + +
+ +
+ ++
+
+
+ +
+ +
+
+ +
Leukocytes Human Porcine Porcine with TF TF-activityb) activity")
+
+
Serum Human
+
+
+ +
+ + + + +
+
+
+
+ + +
+
+ + + + +
+ +
Lympho id tissues Porcine Bovine
+ +
+ +
+
+ + + + +
+
+
Ovine
Sources of dialysates
+ +
+ +
+ + +
+ + + +
+
+ + +
+ + + + + + + +
+
+ +
+
+
+ + +
Non-lym phoid tissues Human Porc Porcine ine Bovine
.) A) orcinol positive a) positive and absorbing 254 nm light with with or without emitting emitting at 366 nm light, B) orcinol orcinol negative, but but absorbing 254 nm light with or without emitting at 366 nm light, C) orcino orcinoll positive only, only, D) ninhydrin positive, E) at 366 nm light emittance emittanc e only. b) Open spaces = no detectable spots.
Localization in G-25 fractions fract ions
Spot Spot number in G-IO fractions fractions
Table 2. Occurre nce of the components in Sephadex G-10 Table G-IO fractions VIa, VIb, Vlb, and VII of and Sephadex G-25 G-2S fractions 5--6 S-f> and in 7 dialysates from leukocyt e, serum, lymphoi d and non serum, non-lym -lympho phoid id organs
:> >
f;:~
§::l0
C c::
:>-
Z! ~
00 00
~
...'-"en
not found not found not found 4 4 not found 4 s-6 5-6 4 not found not not found not found 5~ 5-6 5-6 5-6 not found not not found not found not not found not not found not found not found not not found not not found 8 8 8 8 8 8 8 8 8
IV IV!1 IV 2 IV 33 IV44 IV IVss IV 66 IV 7 IVa IVs V! VI V2 V V33 V4 V Vss V V66 V7 Vs VB V V99 VIII! VIllI VIII 2 VIII)3 VIII VIII VIII44 VIlIs VIII VIII66 IX IX!1 IX IX2 IX IX33 IX44 IX IX IXss IX IX66 IX IX7 IX IXsB
a) See Table 2. .)
I~
Localization G-25 in G -25 fractions fract ions
Spot number in G-I G-IO O fractions
Tryptophan
Guanosine
Tyrosine Inosine
Ribose Ribose Thymidine
Uridine
Phenylalanine
Component identical identical with with reference substances E C B D A A A C A A D B D A B B A B A A C A A A A A A D D D D A
Staining Staining behaviour') behaviour")
leukocyte, serum, lymphoid and non-lymphoid organs organs
+ + + + + + + + +
+
+
+ + +
+ + + + + +
+
+ + + + + + + + + + +
+
+ + +
+
+ +
+ + +
+ + + ++
++ ++ +
+
+ + + + + + ++ +
+ ++ ++ ++
+ + + +
+
Lymphoid tissues Bovine Porcine Bovine
+ +
+
+
Serum Serum Human
+ + + + +
+
+ +
++ + + +
Leukocyt es Leukocytes Human Porcine TFwith TFactivityb) acriviry")
+
+ + + +
+ + +
+
+ + +
+ + + ++
+ + +
+
++
+ +
+
++
++ ++
++
+
+ + ++ + + + + + + ++
+ + + + +
+
+
+
+ + + + ++
+
+
+
+ +
+ + +
+ +
+ + + +
Non-lymphoid tissues Human Porcine Bovine
+
Ovine
Sources Sources of dialysates
'"3
.... V> '" -0 '"
n
..08...
'Tl '11
..
;:t' ...
'"
OJ :I :> II>
'"
...j ...>-1
0 ..., .....
:> :I
~ s: o·
N
f; fti ::1 :! .
n
.'"
n::r o:T "...'"
e!-
n' e,
n::r o:T
ARJA UOTILA 360 . ARJA
A comprehensive comparison of the substances found in various dialysates, after fractionation with Sephadex and TLC is given in tables 2 and 3. None of the fractions tested had components specific for human leukocyte dialysate. However, several components in VIa, VIb and VII were or nearly all (thymine, nicotinamide, niacin, common to all (uracil, VIb 4) or or organ dialysates. VIb 6, VIb 7, glucose, hypoxanthine) cell or Human serum dialysate contained only a few substances in area corresponding to fraction IV-IX. The strongest spot in TLC represented glucose and this material was found in several fractions. Phenylalanine and tyrosine were seen in fractions IV and V, respectively. An unidentified spot in fractions VIa, VIb and VII of human serum dialysate corresponds to VIb 4 (table 2).
Acid hydrolysis 5--6 or or 7 following acid No change was seen in fraction VIa, VIb, VII, 5-6 hydrolyses except the decomposition of uracil and hypoxanthine. No new ninhydrin positive spots were seen, suggesting the absence of detectable amounts of peptides in these fractions.
Discussion In this work we have compared the chemical composition of human d) and other mammalian organ dialysates in order to leukocyte dialysate (TF (TFd) characterize the component(s) responsible for their in vivo effect on deleayed hypersensitivity in man or in guinea pig. The rationale of this or approach is based on the assumption that biologic similarity (16) or differences of the various dialysate would also reflect in their chemical composition. This approach is limited, however, by the fact that all dialysates so far tested (human leukocyte, porcine spleen and bovine liver dialysates) were active in vivo and no proven inactive preparations were available. Human serum dialysate was therefore used as a putative inactive preparation. While it is established that serum is inactive in transferring cellular immunity (18), the effect of serum or specific cellular or serum dialysate in the guinea pig model has not been tested. In this study the chemical analysis was restricted to the Sephadex G-I0 and Sephadex G-25 fractions which eluted at or or after the Vt of the columns, since most investigators have located their in vivo TF d activity of human leukocyte dialysate in both human recipients and antigen primed guinea pig, in this elution region. In this region both gels operate mainly on the adsorption principle, and the order of elution of identified as well as unidentified components are the same in both gels (table 2, 3), and thus results obtained with sephadex G-I0 and G-25 are most probably comparable. The study yielded no firm evidence indicating which component(s) were responsible for the augmenting effect in human leukocyte dialysate or or
361 Chemical Characterization of Transfer Factor . 361
in the guinea pig model with organ dialysates. The uracil containing fraction VIa and the hypoxanthine containing fraction VII as well as fraction VIb, which eluted in between these two larger peaks, contained several unidentified substances common to all or or nearly all dialysates (table 2). In contrast, or in human plasma dialysate contained hardly any spots in these fractions or geOl~ral. This could indicate that some of the the fraction eluting after Vt in general, dialy~ate were responsible for the in vivo effect substances missing in serum dialysate of human leukocyte dialysate in man. None of the spots in fractions VIa, VIb, VII were specific for human leukocyte dialysate, or for the two active organ dialysates derived from porcine spleen and bovine liver. The nature of the unidentified substances in the human leukocyte and unclear. In contrast to the findings of others mammalian organ dialysates is unclear. or mono/oligonucleotides mon%ligonucleotides were not found in the fractions (19, 20), peptides or TFdd activity (VI-VII). However, the present results with the known in vivo TF TF d are in accordance with those of Wilson et al. (11), who found that the TFd activity eluted with hypoxanthine in Sephadex G-25 chromatography. The active fraction, TF x ' which could be further separated from hypoxanthine (11), contained six unidentified components showing similar staining behaviour on TLC as the unidentified components found in the present work in Sephadex G-25 fractions 5-6 and 7. The presence of nicotinamide or close to the TFd's in vivo (21) and inosine (22) in fractions eluting at or activity was also confirmed in the present work. cqaracters in TLC thus support Sephadex chromatography and staining characters the view that the unidentified substances in fractions VIa, VIb, and VII from Sephadex G-l0 and in fractions 5-6 and 7 from Sephadex G-25 were similar to the substances tentatively identified in the present work, as nucleobases, nucleosides and related heterocyclic compounds. In addition, the comparison between the organ dialysates revealed that there were no components specific for human leukocyte dialysate or specific for human leukocyte, porcine spleen and bovine liver dialysate, all of which were capable in augmenting the skin reactivity of the guinea pigs and most of the identified and unidentified substances in in vivo active fractions of human leukocyte dialysate were common to all or nearly all organ dialysates. Although such small molecular components as hypoxanthine and nicotinamide are known to effect lymphocyte functions in vivo (21,23), it dremains to be studied whether or not they are related to the in vivo TF dactivity.
Acknowledgements from Sigrid Juselius Juselius Foundation and Emil The work has been supported with grants from Aaltonen Foundation. The skilled assistance of Mr. PEKKA VIRTANEN, Mr. YOSHIO KAvANOVA, Mr. RALPH EUA KYROLA is gratefully acknowledged. ASHORN and Miss EI]A
ARJA VOTILA 362 . AR]A
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