488 TRANSACTIONS
PASSIVE
OF THE ROYAL
SOCIETY OF TROPICAL
TRANSFER OF IMMUNITY OF TRANSFER FACTOR
MEDICINE AND HYGIENE. VOL. 69. No. 5 & 6.1975.
IN HUMAN SCHISTOSOMIASIS MANSONI: ON EARLY ESTABLISHED INFECTIONS
EFFECT
KENNETH S. WARRENl, JOSEPH A. COOKZ, JOHN R. DAVID3 AND PETER JORDAN4 IDepartments of Medicine and Community Health, Case Western Reserve University School of Medicine and University Hospitals, Cleveland, Ohio, U.S.A. 2Research and Control Department, St. Lucia, West Indies, Stafs Member, Rockefeller Foundation. 3Department of Medicine, Harvard Medical School and Robert Breck Brigham Hospital, Boston, Massachusetts, U.S.A. 4Medical Research Council, seconded to Rockefeller Foundation; Research and Control Department, St. Lucia, West Indies.
Introduction Previous attempts to effect the passivetransfer of humoral immunity to Schistosoma mansoniinfection in man via hyperimmune antischistosomegammaglobulin have been unsuccessful(WARRENet al., 1972; COOKet al., 1972). Accordingly, in the present study an attempt was made to transfer cellular immunity using transfer factor obtained by freezing and thawing human white blood cells and dialyzing the extract. The dialysate obtained from cells from a person exhibiting delayed hypersensitivity will transfer this hypersensitivity to a negative recipient as first described by LAWRENCE, et al., (1963). Transfer factor has been used by investigators in attempts to restore cellular hypersensitivity and immunity in a number of clinical conditions where an immunodeficiency state exists (ROCKLIN et al., 1970a; LEVIN et al., 1970; BULLOCK, et al., 1972; SCHULKIND et al., 1972). In the present study, transfer factor was prepared from leucocytesof adult patients with S. mansonischistosomiasis who exhibited large delayed skin reactions to S. mansoni antigens but showedno signsor symptomsof disease.This transfer factor wascarefully administeredin a hospital setting to children with early and light S. mansoni infections and to uninfected children ascontrols.
Materials
and methods
Donors
Blood for the production of transfer factor was obtained from 9 bananaplantation workers who had strong delayed skin reactions to the adult S. mansoniantigen (TABLE I). General medical evaluations showed chronic asymptomatic schistosomiasis without liver or spleen enlargement in each subject. S. mansoni egg excretions ranged from 10 to 415 eggs/ml. of faeces.No abnormalitieswere found in routine blood or urine tests, and sera from all donors were Australia antigen negative. Donors were also skin tested with PPD, Histoplasma, Brucella, Can&da and mumps antigens. Production
of transfer factor
500 ml. of blood was drawn from each donor into a Fenwal bag, using an ion-exchangeresin as anticoagulant (LAWRENCE and AL-ASKARI, 1971).50 ml. of Macrodex (Dextran 70 from PharmaciaLaboratories, Inc.) was added to eachbag and the red cells were allowed to sedimentin a 37°C. incubator. After 45-60 minutes, most of the red cells were drained from the bottom of the bag and discarded.The buffy coat-rich plasmawas collected in 50 ml. sterile centrifuge tubes and spun for 10 minutes at 1,200r.p.m. The supernatant plasmawas discarded and the sedimentscontaining white cells and some red cells were pooled in a 15ml. centrifuge tube. After this, the tube was spun for 10 minutes at 800 r.p.m., the white cell layer was measured,an equal amount of distilled water was added, and the mixture was frozen and stored at -40°C.
These investigations have been supported by the Rockefeller Foundation. Reprint requests may be addressed to Dr. K. S. Warren, Department of Medicine, Building, University Hospitals, Cleveland, Ohio 44106, U.S.A
Wearn
Research
KENNETH
S. WARREN,
JOSEPH
A. COOK,
JOHN
R. DAVID
AND
PETER
489
JORDAN
TABLEI. Donors of transfer factor. Skin Test * cm2 Donors
Sex/Age
Mean eggoutput eggs/ml.faeces
15 min.
24 hr.
CL
M/21
160
5.7
5.4
PM
M/28
415
3.9
2-l
RI
M/37
54
2.5
1.9
GG
M/38
184
2.5
2-l
AMs
M/46
49
4.1
3.7
EMS
M/53
106
1.9
3.9
EM
M/60
125
3.7
2.8
EN
M/65
28
1.8
3-o
JC
M/45
10
5-o
2.7
*WHO standardadult S. munsoniantigen 35 mg./ml. protein. The frozen white blood cells were thawed and frozen 7-10 times using a dry ice-acetonemixture and then were incubated with 1 mg. DNAse (Worthington Biochemicals,Inc.) in the presenceof magnesium ions for 30 minutes at 37°C. The resultant cell extract was dialyzed against 100 ml. distilled water for 24 hours in a cold room at 4°C. The dialysate was lyophilized in 50 ml. aliquots. Each dose (equivalent to 1 ml. of packed white cells) was dissolvedin 1.7 ml. pyrogen-free distilled water and sterilized by passage through a Millipore filter (0.45 microns) before use. Recipients
Recipients of transfer factor were selectedby examining the records of the annual quantitative stool examinations carried out on a large segmentof the local population over a period of 4 years. 8 children were selectedwho were negative for 2 to 3 examinationsand first becamepositive at a light intensity of infection (lessthan 100 eggs/ml. faeces)3 months before the present experiments began. After obtaining parental consent, the children were admitted to the medical ward of the Researchand Control Department, where histories were taken and physical examinationsand laboratory testswere performed. The laboratory tests included haematocrit, white blood count and differential, total protein, albumin, globulin and serum glutamic oxaloacetic transaminase;these were repeated at least 5 times during the 5-week course of the study. The children all had intestinal helminth infections, eosinophilia, and mild microcytic anaemia. Daily determinations during the stay in hospital included body weight, temperature and quantitative’ studies of stoolsfor parasite eggsusing a modification of the Bell technique (COOK and JORDAN, 1971). The first child treated was the most heavily infected (T.G.); he received multiple small dosesof transfer factor while being monitored carefully for adverse signs, symptoms and changesin laboratory tests. Since he showedno local reaction, no fever, no enlargementof liver or spleenand no changesin white counts and differentials, or liver function tests, we instituted treatment of the other children using the following regimen: after 2 weeks in hospital, each child received a dose of transfer factor (equivalent to 1 ml. packed white cells) in 1.7 ml. pyrogen-free water injected into the deltoid of both arms, half of the doseintradermally and the other half subcutaneously. 10 days to 2 weeks later they were treated in the samemanner with another dose of transfer factor. 2 of 8 children were randomly selectedas controls and were treated with salineinjections. None of the children showedany ill effects of the treatment. The children were skin tested with PPD, Histoplasma, Brucella, Can&da and mumps antigensbefore the first dose of transfer factor. They were retested with the above antigens and also adult S. mansoni antigen 9 days after the first doseof transfer factor and a similar period of time after the second dose (a reaction was consideredpositive to S. mansoni when it was 1-Ocm2 or greater). They were not skin tested with S. mansoni antigen prior to receiving transfer factor since previous studies had shown
PASSIVETRANSFEROFIMMUNITYINHUMAN
490
TABLE II. Infected
SCHISTOSOMIASISMANSO~
recipients
of transfer factor. Hospital
Field studies Last negative stool
Eggs/ml. faeces April 1972
Eggs/ml. Before
Recipients of Transfer Factor AN
F/7
AJP
F/10
PJ
M/10
TG
M/11
1971
30
21 f
3.2
P21 1971
30
9 i.
2.4
I.141
M/9
Ml7
Recipients
of saline
MH
Fill
1970
90
285 rt 40
1971
60
35 +
6-2
1970
60
105 f
15.6
P21
Skin test** cm2 15 min.
24 hr.
1971
10
17 rt
M/12
1971
10
38 i:
[I31 *Number of determinations shown in [ 1. **WHO standard adult S. mansuni antigen 35 mg. protein/ml., factor. Figures in ( ) are for 6 month follow up.
33 f
4.5
Md 6 i
1.1
I31 286 f
43
WI 24 &
4.2
[W 64 31 15.0
P71 2.5
WI MF
1972
Sex/Age
1131 SC
faeces* After
1 71 RF
studies-August
15 f
2.5
I.171 8.2
31 &
P21 administered
(:::)
3.4
(: 103)
("0::)
9 days after initial dose of transfer
that only 35% of infected children had delayed reactions, most of which were borderline positive, the mean lesion area being O-7 cm2 (WARRENet al., 1973). Furthermore, it was feared that the injection of antigen itself might induce a positive skin test. Fluorescent antibody determinations were performed before and after treatment as described previously (SADUN, ANDERSON and WILLIAMS, 1962). Follow up studies, including skin test reactivity and 24 hour faecal egg output, were carried out in hospital for 3-day periods at 1,3 and 6 months after the final doseof transfer factor. In addition, 12 children, ageand sex matched to the above but uninfected on the basisof 3 negative stool examinations,were alsostudied. 6 received 2 dosesof transfer factor from the samedonors as above and 6 received saline. Antigen-induced aH-thymidine incorporation &to lymphocytes 20 ml. of venous blood was drawn; heparin was used as anticoagulant. The red blood cells were removed by sedimentation with dextran and the huffy coat centrifuged on Ficoll-Hypaque (BOYUM, 1968). The mononuclear-rich fraction was washedand a concentration preparedof 1 x lo6 cells per ml.
of minimal Eagle’smedium supplementedwith glutamine and 15% foetal calf serum. WHO reference skin test antigen (Melcher’s adult Schistosoma mansoni) was dialyzed with 5 changes in salineto remove preservative, and a 1 : 10 dilution wasmade. One-tenth of either undiluted or diluted antigen was added per ml. of cell suspensionand the material cultured at 37°C. under 5% CO, and air atmosphere.On the fifth day 1 PC of 3H-thymidine wasadded to the cultures. 24 hours later, the cultures were harvested, washedwith saline,and the cellstreated with trichloracetic acid and methanol aspreviously described(ROCKLIN et al., 1970b).Radioactivity wasassessed in a scintillation counter. In addition, Can&u and streptokinase-streptodomaseantigens were used as controls.
KENNETH
S. WARREN,
JOSEPH
A. COOK,
TABLE III. Non-infected Recipients of
JOHN
R. DAVID
AND
PETER
recipients of transfer factor. Skin test to S. mansoni
Sex/Age
15 min. Transfer factor VH
491
JORDAN
24 hr.
F/7
1.9
0.5
AV
F/10
0.7
0.7
AR
M/10
O-5
o-0
PSM
M/11
0.4
o-0
SH
M/9
0.3
1.4
FE
Ml7
1.5
070
Saline: ss
F/7
0.2
0.3
FH
F/l0
0.2
o-0
CE
M/10
0.6
o-o
DA
M/11
l-2
0.4
CM
Ml9
0.8
1.1
CA
Ml7
0.6
0.7
TABLEIV. S. mansoni antigen-induced SH-thymidine incorporation into lymphocytes*. Recipientst
Stimulation index
Control counts per minute.
S. mansoni
SK-SD or Candida
AN
1205
3.6
11.3
AJP
5985
0.8
3.1
PJ
4461
3.3
8*1
TG
448
3.6
3.6
RF
982
1.3
13.3
SC
13,162
0.9
2.9
MH
5160
3.3
3.6
MF
8147
1.7
3-l
*Lymphocyte cultures were carried out in duplicate. Concentration of antigen: 0.1 ml. of l/10 dilution of dialyzed S. mansoni skin test material/ml. cell suspension;50 units SK-SD/ml cell suspension. -tThe first 6 subjectsreceived Transfer Factor., and the last 2 received saline. Results Six of eight children with mild S. mansoni infections received 2 dosesof transfer factor obtained from donors who exhibited intense cutaneous delayed hypersensitivity reactions to WHO S. mansoniantigen; 2 children received saline. As can be seenin Table II, only 1 of the children (T.G.) exhibited a positive
492
PASSIVE
TRANSFER
OF IMMUNITY
IN HUMAN
SCHISTOSOMIASIS
MANSONI
TABLE V. In vitro cultures using various media*.
Culture conditions Serum
Medium
Control counts per minute
Stimulation index S. mansoni
SK-SD
Autologous
199-Hepes
589
17.0
95.6
FBS
199-Hepes
1313
1.7
4.8
FBS
MEM-CO,
1637
-
25.3
#Lymphocytes were cultured for 6 days. S. mansoniwas a salineextract of adult wormsand wasadded,so final concentration was 5 pg/ml. No CO, was added to TCM 199-Hepes, but was present with Eagle’s
medium.
delayed hypersensitivity skin test to S. mansoni,after receiving transfer factor. Upon retesting 6 months later, an additional patient exhibited a positive reaction. There was no difference in the faecal egg output before or after treatment with transfer factor, nor was there any difference in other laboratory tests. 6 of 12 children who were not infected with S. mansonialso received transfer factor from the same donors, and 6 others were given saline. As can be seen in Table III, one child in each group exhibited a positive delayed skin reaction to S. mansuni. It should be noted that both infected and non-infected children exhibited normal delayed hypersensitivity to other bacterial antigens; none was anergic (data not shown). Antigen-induced 3H-thymidine incorporation was evaluated before and after transfer factor. Due to unfortunate problems with CO,, most of the first set of cultures were lost. The data from the cultures set up after transfer factor therapy but just prior to skin testing are shown in Table IV. 3 of the treated group and 1 of the control showed a positive stimulation index to S. mansoni (counts in S. mansonilcountsin control > 2). One treated child had a positive skin test when tested 6 months later. The other 2 children whose cells gave positive in vitro results were skin test negative to S. mansoni. In addition, the in vitro assay on cells from one of the donors of transfer factor, whose skin test wasmarkedly positive, was negative. On the other hand, all children were skin test-positive either to SK-SD or Can&da antigen, and their cells showed positive stimulation indices to the appropriate antigen. Because of the initial problem encountered with obtaining enough 5% CO, to maintain the pH of the cultures, an additional experiment was carried out in which lymphocytes were cultured in either autologous serum or foetal calf serum, using Tissue Culture Medium 199 buffered with Hepes Solution; no CO, was.used in the incubation with this medium. The results are shown in Table V. These are obviously preliminary studies and more in vitro assaysneed to be carried out on both infected and normal subjects using S. mansoniantigens. They do indicate, however, that S. mansoni antigen can be used to stimulate human lymphocytes in vitro, that such stimulation can be accomplished using TCM 199 with Hepes ‘buffer, which is better for field work as no CO, is required, and that stimulation is greater when carried out in the presence of human serum than in foetal calf serum. Discussion
The results given here show that our attempt to effect the passive transfer of cell-mediated immunity to S. mansoni infection with transfer factor was unsuccessful. Delayed hypersensitivity may have been transferred in 1 of 6 infected children treated with transfer factor, but no change in egg output was noted. Although this child may have had a positive skin test prior to treatment, the very large size of the skin reaction is highly unusual in children of this age (WARREN et al., 1973). To test whether or not failure to transfer delayed hypersensitivity in these infected children might have been caused by a humoral blocking factor, 6 normal uninfected children were given transfer factor and another 6 were given saline; only 1 in each group exhibited a positive skin test. No change in egg output was observed after treatment in any of the 6 infected children, suggesting that transfer factor had no effect on the worms.
KENNETH
S. WARREN,
JOSEPH A. COOK,
JOHN R. DAVID
AND PETER JORDAN
493
Although it is possible that the transfer factor used in these studies may have been inactive, it was obtained from donors exhibiting strong delayed skin test reactions to S. mansoni antigens by experienced investigators who had previously obtained active transfer factor. While gross delayed skin reactions were not observed, it is possible that there was local infiltration of mononuclear cells detectable only by skin biopsy, as recently described in studies on monkeys by MADDISON et al. (1972). Our attemps to transfer immunity to S. mansoni in man with either hyperimmune gamma globulin or transfer factor have been uniformly negative. Although these studies were performed with great care, it is still possiblethat the reagentswere not potent enough, the dosagewas not high enough, or the timing of administration was not correct. Combined therapy might be more effective. Finally, a significant degreeof immunity in terms of resistanceto infection may not be present in most individuals infected with S. mansoni (WARREN, 1973), particularly if the infections are of low intensity as is common on the island of St. Lucia. Acknowledgment We would like to thank Dr. H. Sherwood Lawrence for his very helpful discussionsconcerning the protocol of thesestudies and the collection of transfer factor; the nursing staff of the researchward of the hospital for their aid in contacting and caring for the children; and Lilian Woodstock, Michael Katz, Le Ming Hang, Pierre A. Peters and Roberta David for their invaluable technical assistance. References BOYUM, A. (1968). Stand. BULLOCK, W. E., FIELDS, COOK, J. A. & JORDAN, I’.
J. clin. Lab. Invest. Suppl. 97, 21, 7. J. I?. & BRANDRISS, M. W. (1972). New Engl. J. Med., 287 1053. (1971). Am. J. trop. Med. Hyg., 20, 84. -, WARREN, K. S. & JORDAN, I’. (1972). Trans. R. Sot. trop. Med. Hyg., 66,777. LAWRENCE, H. S. & AL-ASKARI, S. (1971). In: In Vitro Methods in Cell-Mediated Immunity, p. 531. London: Academic Press. -, -, DAVID, J. R., FRANKLIN, E. C. & ZWEIMAN, B. (1963). Trans. Ass. Am. Physns., 76, 84. , LEVIN, A. S., SPITLER, L. E., STITES, D. I’. & FUDENBERG, H. H. (1970). Proc. natn. Acad. Sci., U.S.A., 67, 821. MADDISON, S. E., HICXLIN, M. D., CONWAY, B. P. & KAGAN, I. G. (1972). Science, 178,757. ROCKLIN, R. E., CHILGREN, R. A., HONG, R. & DAVID, J. R. (1970a). Cell. ImmunoZ., 1, 290. -, REARDON, G., SHEFFER, A., CHURCHILL, W. H. & DAVID, J. R. (1970b). In: Proc. Fifth Leucocyte Culture Conf., p. 639. New York: Academic Press. SADUN, E. H., ANDERSON, R. I. & WILLIAMS, J. S. (1962). Bull. WZd Hlth Org., 27, 151. SCHULKIND, M. L., ADLER, W. H., III, ALTEMEIER, W. A., III & WYOUB, E. M. (1972). Cell. Immunol., 3, 606. WARREN, K. S. (1973). J. infect. Dis., 127, 595. -, COOK, J. A. & JORDAN, P. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 65. -, KELLERMEYER, R. W., JORDAN, I?., LITTELL, A. S., COOK, J. A. & KAGAN, I. G. (1973). Am. J. trop. Med. Hyg., 22, 189.
PASSIVE
OF THE ROYAL
SOCIETY OF TROPICAL
TRANSFER OF IMMUNITY OF TRANSFER FACTOR
MEDICINE AND HYGIENE. VOL. 69. No. 5 & 6.1975.
IN HUMAN SCHISTOSOMIASIS MANSONI: ON EARLY ESTABLISHED INFECTIONS
EFFECT
KENNETH S. WARRENl, JOSEPH A. COOKZ, JOHN R. DAVID3 AND PETER JORDAN4 IDepartments of Medicine and Community Health, Case Western Reserve University School of Medicine and University Hospitals, Cleveland, Ohio, U.S.A. 2Research and Control Department, St. Lucia, West Indies, Stafs Member, Rockefeller Foundation. 3Department of Medicine, Harvard Medical School and Robert Breck Brigham Hospital, Boston, Massachusetts, U.S.A. 4Medical Research Council, seconded to Rockefeller Foundation; Research and Control Department, St. Lucia, West Indies.
Introduction Previous attempts to effect the passivetransfer of humoral immunity to Schistosoma mansoniinfection in man via hyperimmune antischistosomegammaglobulin have been unsuccessful(WARRENet al., 1972; COOKet al., 1972). Accordingly, in the present study an attempt was made to transfer cellular immunity using transfer factor obtained by freezing and thawing human white blood cells and dialyzing the extract. The dialysate obtained from cells from a person exhibiting delayed hypersensitivity will transfer this hypersensitivity to a negative recipient as first described by LAWRENCE, et al., (1963). Transfer factor has been used by investigators in attempts to restore cellular hypersensitivity and immunity in a number of clinical conditions where an immunodeficiency state exists (ROCKLIN et al., 1970a; LEVIN et al., 1970; BULLOCK, et al., 1972; SCHULKIND et al., 1972). In the present study, transfer factor was prepared from leucocytesof adult patients with S. mansonischistosomiasis who exhibited large delayed skin reactions to S. mansoni antigens but showedno signsor symptomsof disease.This transfer factor wascarefully administeredin a hospital setting to children with early and light S. mansoni infections and to uninfected children ascontrols.
Materials
and methods
Donors
Blood for the production of transfer factor was obtained from 9 bananaplantation workers who had strong delayed skin reactions to the adult S. mansoniantigen (TABLE I). General medical evaluations showed chronic asymptomatic schistosomiasis without liver or spleen enlargement in each subject. S. mansoni egg excretions ranged from 10 to 415 eggs/ml. of faeces.No abnormalitieswere found in routine blood or urine tests, and sera from all donors were Australia antigen negative. Donors were also skin tested with PPD, Histoplasma, Brucella, Can&da and mumps antigens. Production
of transfer factor
500 ml. of blood was drawn from each donor into a Fenwal bag, using an ion-exchangeresin as anticoagulant (LAWRENCE and AL-ASKARI, 1971).50 ml. of Macrodex (Dextran 70 from PharmaciaLaboratories, Inc.) was added to eachbag and the red cells were allowed to sedimentin a 37°C. incubator. After 45-60 minutes, most of the red cells were drained from the bottom of the bag and discarded.The buffy coat-rich plasmawas collected in 50 ml. sterile centrifuge tubes and spun for 10 minutes at 1,200r.p.m. The supernatant plasmawas discarded and the sedimentscontaining white cells and some red cells were pooled in a 15ml. centrifuge tube. After this, the tube was spun for 10 minutes at 800 r.p.m., the white cell layer was measured,an equal amount of distilled water was added, and the mixture was frozen and stored at -40°C.
These investigations have been supported by the Rockefeller Foundation. Reprint requests may be addressed to Dr. K. S. Warren, Department of Medicine, Building, University Hospitals, Cleveland, Ohio 44106, U.S.A
Wearn
Research
KENNETH
S. WARREN,
JOSEPH
A. COOK,
JOHN
R. DAVID
AND
PETER
489
JORDAN
TABLEI. Donors of transfer factor. Skin Test * cm2 Donors
Sex/Age
Mean eggoutput eggs/ml.faeces
15 min.
24 hr.
CL
M/21
160
5.7
5.4
PM
M/28
415
3.9
2-l
RI
M/37
54
2.5
1.9
GG
M/38
184
2.5
2-l
AMs
M/46
49
4.1
3.7
EMS
M/53
106
1.9
3.9
EM
M/60
125
3.7
2.8
EN
M/65
28
1.8
3-o
JC
M/45
10
5-o
2.7
*WHO standardadult S. munsoniantigen 35 mg./ml. protein. The frozen white blood cells were thawed and frozen 7-10 times using a dry ice-acetonemixture and then were incubated with 1 mg. DNAse (Worthington Biochemicals,Inc.) in the presenceof magnesium ions for 30 minutes at 37°C. The resultant cell extract was dialyzed against 100 ml. distilled water for 24 hours in a cold room at 4°C. The dialysate was lyophilized in 50 ml. aliquots. Each dose (equivalent to 1 ml. of packed white cells) was dissolvedin 1.7 ml. pyrogen-free distilled water and sterilized by passage through a Millipore filter (0.45 microns) before use. Recipients
Recipients of transfer factor were selectedby examining the records of the annual quantitative stool examinations carried out on a large segmentof the local population over a period of 4 years. 8 children were selectedwho were negative for 2 to 3 examinationsand first becamepositive at a light intensity of infection (lessthan 100 eggs/ml. faeces)3 months before the present experiments began. After obtaining parental consent, the children were admitted to the medical ward of the Researchand Control Department, where histories were taken and physical examinationsand laboratory testswere performed. The laboratory tests included haematocrit, white blood count and differential, total protein, albumin, globulin and serum glutamic oxaloacetic transaminase;these were repeated at least 5 times during the 5-week course of the study. The children all had intestinal helminth infections, eosinophilia, and mild microcytic anaemia. Daily determinations during the stay in hospital included body weight, temperature and quantitative’ studies of stoolsfor parasite eggsusing a modification of the Bell technique (COOK and JORDAN, 1971). The first child treated was the most heavily infected (T.G.); he received multiple small dosesof transfer factor while being monitored carefully for adverse signs, symptoms and changesin laboratory tests. Since he showedno local reaction, no fever, no enlargementof liver or spleenand no changesin white counts and differentials, or liver function tests, we instituted treatment of the other children using the following regimen: after 2 weeks in hospital, each child received a dose of transfer factor (equivalent to 1 ml. packed white cells) in 1.7 ml. pyrogen-free water injected into the deltoid of both arms, half of the doseintradermally and the other half subcutaneously. 10 days to 2 weeks later they were treated in the samemanner with another dose of transfer factor. 2 of 8 children were randomly selectedas controls and were treated with salineinjections. None of the children showedany ill effects of the treatment. The children were skin tested with PPD, Histoplasma, Brucella, Can&da and mumps antigensbefore the first dose of transfer factor. They were retested with the above antigens and also adult S. mansoni antigen 9 days after the first doseof transfer factor and a similar period of time after the second dose (a reaction was consideredpositive to S. mansoni when it was 1-Ocm2 or greater). They were not skin tested with S. mansoni antigen prior to receiving transfer factor since previous studies had shown
PASSIVETRANSFEROFIMMUNITYINHUMAN
490
TABLE II. Infected
SCHISTOSOMIASISMANSO~
recipients
of transfer factor. Hospital
Field studies Last negative stool
Eggs/ml. faeces April 1972
Eggs/ml. Before
Recipients of Transfer Factor AN
F/7
AJP
F/10
PJ
M/10
TG
M/11
1971
30
21 f
3.2
P21 1971
30
9 i.
2.4
I.141
M/9
Ml7
Recipients
of saline
MH
Fill
1970
90
285 rt 40
1971
60
35 +
6-2
1970
60
105 f
15.6
P21
Skin test** cm2 15 min.
24 hr.
1971
10
17 rt
M/12
1971
10
38 i:
[I31 *Number of determinations shown in [ 1. **WHO standard adult S. mansuni antigen 35 mg. protein/ml., factor. Figures in ( ) are for 6 month follow up.
33 f
4.5
Md 6 i
1.1
I31 286 f
43
WI 24 &
4.2
[W 64 31 15.0
P71 2.5
WI MF
1972
Sex/Age
1131 SC
faeces* After
1 71 RF
studies-August
15 f
2.5
I.171 8.2
31 &
P21 administered
(:::)
3.4
(: 103)
("0::)
9 days after initial dose of transfer
that only 35% of infected children had delayed reactions, most of which were borderline positive, the mean lesion area being O-7 cm2 (WARRENet al., 1973). Furthermore, it was feared that the injection of antigen itself might induce a positive skin test. Fluorescent antibody determinations were performed before and after treatment as described previously (SADUN, ANDERSON and WILLIAMS, 1962). Follow up studies, including skin test reactivity and 24 hour faecal egg output, were carried out in hospital for 3-day periods at 1,3 and 6 months after the final doseof transfer factor. In addition, 12 children, ageand sex matched to the above but uninfected on the basisof 3 negative stool examinations,were alsostudied. 6 received 2 dosesof transfer factor from the samedonors as above and 6 received saline. Antigen-induced aH-thymidine incorporation &to lymphocytes 20 ml. of venous blood was drawn; heparin was used as anticoagulant. The red blood cells were removed by sedimentation with dextran and the huffy coat centrifuged on Ficoll-Hypaque (BOYUM, 1968). The mononuclear-rich fraction was washedand a concentration preparedof 1 x lo6 cells per ml.
of minimal Eagle’smedium supplementedwith glutamine and 15% foetal calf serum. WHO reference skin test antigen (Melcher’s adult Schistosoma mansoni) was dialyzed with 5 changes in salineto remove preservative, and a 1 : 10 dilution wasmade. One-tenth of either undiluted or diluted antigen was added per ml. of cell suspensionand the material cultured at 37°C. under 5% CO, and air atmosphere.On the fifth day 1 PC of 3H-thymidine wasadded to the cultures. 24 hours later, the cultures were harvested, washedwith saline,and the cellstreated with trichloracetic acid and methanol aspreviously described(ROCKLIN et al., 1970b).Radioactivity wasassessed in a scintillation counter. In addition, Can&u and streptokinase-streptodomaseantigens were used as controls.
KENNETH
S. WARREN,
JOSEPH
A. COOK,
TABLE III. Non-infected Recipients of
JOHN
R. DAVID
AND
PETER
recipients of transfer factor. Skin test to S. mansoni
Sex/Age
15 min. Transfer factor VH
491
JORDAN
24 hr.
F/7
1.9
0.5
AV
F/10
0.7
0.7
AR
M/10
O-5
o-0
PSM
M/11
0.4
o-0
SH
M/9
0.3
1.4
FE
Ml7
1.5
070
Saline: ss
F/7
0.2
0.3
FH
F/l0
0.2
o-0
CE
M/10
0.6
o-o
DA
M/11
l-2
0.4
CM
Ml9
0.8
1.1
CA
Ml7
0.6
0.7
TABLEIV. S. mansoni antigen-induced SH-thymidine incorporation into lymphocytes*. Recipientst
Stimulation index
Control counts per minute.
S. mansoni
SK-SD or Candida
AN
1205
3.6
11.3
AJP
5985
0.8
3.1
PJ
4461
3.3
8*1
TG
448
3.6
3.6
RF
982
1.3
13.3
SC
13,162
0.9
2.9
MH
5160
3.3
3.6
MF
8147
1.7
3-l
*Lymphocyte cultures were carried out in duplicate. Concentration of antigen: 0.1 ml. of l/10 dilution of dialyzed S. mansoni skin test material/ml. cell suspension;50 units SK-SD/ml cell suspension. -tThe first 6 subjectsreceived Transfer Factor., and the last 2 received saline. Results Six of eight children with mild S. mansoni infections received 2 dosesof transfer factor obtained from donors who exhibited intense cutaneous delayed hypersensitivity reactions to WHO S. mansoniantigen; 2 children received saline. As can be seenin Table II, only 1 of the children (T.G.) exhibited a positive
492
PASSIVE
TRANSFER
OF IMMUNITY
IN HUMAN
SCHISTOSOMIASIS
MANSONI
TABLE V. In vitro cultures using various media*.
Culture conditions Serum
Medium
Control counts per minute
Stimulation index S. mansoni
SK-SD
Autologous
199-Hepes
589
17.0
95.6
FBS
199-Hepes
1313
1.7
4.8
FBS
MEM-CO,
1637
-
25.3
#Lymphocytes were cultured for 6 days. S. mansoniwas a salineextract of adult wormsand wasadded,so final concentration was 5 pg/ml. No CO, was added to TCM 199-Hepes, but was present with Eagle’s
medium.
delayed hypersensitivity skin test to S. mansoni,after receiving transfer factor. Upon retesting 6 months later, an additional patient exhibited a positive reaction. There was no difference in the faecal egg output before or after treatment with transfer factor, nor was there any difference in other laboratory tests. 6 of 12 children who were not infected with S. mansonialso received transfer factor from the same donors, and 6 others were given saline. As can be seen in Table III, one child in each group exhibited a positive delayed skin reaction to S. mansuni. It should be noted that both infected and non-infected children exhibited normal delayed hypersensitivity to other bacterial antigens; none was anergic (data not shown). Antigen-induced 3H-thymidine incorporation was evaluated before and after transfer factor. Due to unfortunate problems with CO,, most of the first set of cultures were lost. The data from the cultures set up after transfer factor therapy but just prior to skin testing are shown in Table IV. 3 of the treated group and 1 of the control showed a positive stimulation index to S. mansoni (counts in S. mansonilcountsin control > 2). One treated child had a positive skin test when tested 6 months later. The other 2 children whose cells gave positive in vitro results were skin test negative to S. mansoni. In addition, the in vitro assay on cells from one of the donors of transfer factor, whose skin test wasmarkedly positive, was negative. On the other hand, all children were skin test-positive either to SK-SD or Can&da antigen, and their cells showed positive stimulation indices to the appropriate antigen. Because of the initial problem encountered with obtaining enough 5% CO, to maintain the pH of the cultures, an additional experiment was carried out in which lymphocytes were cultured in either autologous serum or foetal calf serum, using Tissue Culture Medium 199 buffered with Hepes Solution; no CO, was.used in the incubation with this medium. The results are shown in Table V. These are obviously preliminary studies and more in vitro assaysneed to be carried out on both infected and normal subjects using S. mansoniantigens. They do indicate, however, that S. mansoni antigen can be used to stimulate human lymphocytes in vitro, that such stimulation can be accomplished using TCM 199 with Hepes ‘buffer, which is better for field work as no CO, is required, and that stimulation is greater when carried out in the presence of human serum than in foetal calf serum. Discussion
The results given here show that our attempt to effect the passive transfer of cell-mediated immunity to S. mansoni infection with transfer factor was unsuccessful. Delayed hypersensitivity may have been transferred in 1 of 6 infected children treated with transfer factor, but no change in egg output was noted. Although this child may have had a positive skin test prior to treatment, the very large size of the skin reaction is highly unusual in children of this age (WARREN et al., 1973). To test whether or not failure to transfer delayed hypersensitivity in these infected children might have been caused by a humoral blocking factor, 6 normal uninfected children were given transfer factor and another 6 were given saline; only 1 in each group exhibited a positive skin test. No change in egg output was observed after treatment in any of the 6 infected children, suggesting that transfer factor had no effect on the worms.
KENNETH
S. WARREN,
JOSEPH A. COOK,
JOHN R. DAVID
AND PETER JORDAN
493
Although it is possible that the transfer factor used in these studies may have been inactive, it was obtained from donors exhibiting strong delayed skin test reactions to S. mansoni antigens by experienced investigators who had previously obtained active transfer factor. While gross delayed skin reactions were not observed, it is possible that there was local infiltration of mononuclear cells detectable only by skin biopsy, as recently described in studies on monkeys by MADDISON et al. (1972). Our attemps to transfer immunity to S. mansoni in man with either hyperimmune gamma globulin or transfer factor have been uniformly negative. Although these studies were performed with great care, it is still possiblethat the reagentswere not potent enough, the dosagewas not high enough, or the timing of administration was not correct. Combined therapy might be more effective. Finally, a significant degreeof immunity in terms of resistanceto infection may not be present in most individuals infected with S. mansoni (WARREN, 1973), particularly if the infections are of low intensity as is common on the island of St. Lucia. Acknowledgment We would like to thank Dr. H. Sherwood Lawrence for his very helpful discussionsconcerning the protocol of thesestudies and the collection of transfer factor; the nursing staff of the researchward of the hospital for their aid in contacting and caring for the children; and Lilian Woodstock, Michael Katz, Le Ming Hang, Pierre A. Peters and Roberta David for their invaluable technical assistance. References BOYUM, A. (1968). Stand. BULLOCK, W. E., FIELDS, COOK, J. A. & JORDAN, I’.
J. clin. Lab. Invest. Suppl. 97, 21, 7. J. I?. & BRANDRISS, M. W. (1972). New Engl. J. Med., 287 1053. (1971). Am. J. trop. Med. Hyg., 20, 84. -, WARREN, K. S. & JORDAN, I’. (1972). Trans. R. Sot. trop. Med. Hyg., 66,777. LAWRENCE, H. S. & AL-ASKARI, S. (1971). In: In Vitro Methods in Cell-Mediated Immunity, p. 531. London: Academic Press. -, -, DAVID, J. R., FRANKLIN, E. C. & ZWEIMAN, B. (1963). Trans. Ass. Am. Physns., 76, 84. , LEVIN, A. S., SPITLER, L. E., STITES, D. I’. & FUDENBERG, H. H. (1970). Proc. natn. Acad. Sci., U.S.A., 67, 821. MADDISON, S. E., HICXLIN, M. D., CONWAY, B. P. & KAGAN, I. G. (1972). Science, 178,757. ROCKLIN, R. E., CHILGREN, R. A., HONG, R. & DAVID, J. R. (1970a). Cell. ImmunoZ., 1, 290. -, REARDON, G., SHEFFER, A., CHURCHILL, W. H. & DAVID, J. R. (1970b). In: Proc. Fifth Leucocyte Culture Conf., p. 639. New York: Academic Press. SADUN, E. H., ANDERSON, R. I. & WILLIAMS, J. S. (1962). Bull. WZd Hlth Org., 27, 151. SCHULKIND, M. L., ADLER, W. H., III, ALTEMEIER, W. A., III & WYOUB, E. M. (1972). Cell. Immunol., 3, 606. WARREN, K. S. (1973). J. infect. Dis., 127, 595. -, COOK, J. A. & JORDAN, P. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 65. -, KELLERMEYER, R. W., JORDAN, I?., LITTELL, A. S., COOK, J. A. & KAGAN, I. G. (1973). Am. J. trop. Med. Hyg., 22, 189.
