CLINICAL
,MM”NOLOGY
AND
IMMUNOPATHOLOGY
11,
183-190 (1978)
Preliminary Results of Transfer Factor Therapy Persistant Cutaneous Leishmania Infection MANJIT Iranian
K. SHARMA, National
FARVARDIN
Blood
Transfusion
ANARAKI, Service,
of
AND .F. ALA Tehran,
Iran
Received August 10, 1978 Three patients with persistent cutaneous leishmania infection of &, 14- and 30year duration, respectively, and with demonstrable delayed hypersensitivity to leishmania antigens were treated with leishmania-specific transfer factor prepared from healthy donors with a previous history of cutaneous leishmania infection. The cutaneous lesions healed completely in all three cases. The total dose of transfer factor required to achieve healing was much higher than the dose used by other investigators. The results justify a further clinical trial to determine the therapeutic effectiveness of transfer factor in cutaneous leishmaniasis. Further, because the infective agent is neither ubiquitous nor homogeneous, cutaneous leishmania infection in man offers a unique opportunity for a double blind study aimed at evaluating the therapeutic usefulness of transfer factor in human intracellular infections and further analytic studies to determine both the in vivo and in vitro specificity of transfer factor.
INTRODUCTION
Cutaneous leishmania infection (CLI) in man, as it is generally seen, is a selflimiting disease. The cutaneous lesion usually heals within months, although in rare cases, the cutaneous lesion persists for several years (1, 2). The causes of persistent lesions in an otherwise healthy individual are not known. Defects of the host’s immune response have been suggested as one possible explanation (1). Cell-mediated immunity (CMI) appears to play an important role in recovery from CLI, in both man and experimental animals. However, the development of CM1 is not always accompanied by recovery from the infection (3-5). Commonly used antiprotozoal drugs do not appear to change the course of self-limiting cutaneous lesions significantly and they are totally ineffective against persistent CLI. In the past, transfer factor (TFd), a dialyzable extract of leukocytes, has been used with some limited success in the treatment of a number of intracellular infections caused by mycobacteria, viruses, and fungi (6-16). These infections were associated with either normal or impaired CM1 to the infective agent in question. TFd therapy was attempted in three patients with persistent CL1 of several years duration who had failed to respond to antiprotozoal therapy in the past. CASE REPORT Patient I. A 35year-old
male presented with cutaneous leishmania infection of the left shin, persisting for the past 8 years. The skin lesion which was very small initially had progressed to involve most of the lower leg. The lesion was ulcerated and contained yellow cheesy material in several places. The skin adjoining the infected area showed minimal induration. His leishmanin skin test was positive, although 5 years after infection it was reported to be negative. His serum im183 0090-1229/‘79/020183-08$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved
184
SHARhl.4.
ASARAKI,
AND
:\I.:\
munoglobulins and C, levels were normal except for 1gM which was elevated (IgG, 14.2 mg/ml; IgA. 2.8 mg/ml; IgM. 3.4 mg/ml: C:,. 0.6 mgiml). Serum leishmania-specific fluorescent antibody titer was 1: 1024. Leishmania were demonstrable in lesions, by both smear and culture. PNtiettt _3. A 16-year-old male presented in whom cutaneous lesions of the face developed at the age of 2 years. The lesions appeared dry in the center and were surrounded by indurated margins. Leishmania in the infected area were demonstrable by skin biopsy. He was leishmanin-positive. His serum immunoglobulins were normal (IgG. 6.7 mg/ml: IgA. 3.2 mgiml: 1gM. 1.6 mgiml), and C., was low normal (0.5 mg/ml). Patient 3. A 33-year-old female presented in whom cutaneous lesions of the face developed at the age of 3 years. The lesions generally remained dry; however. from time to time they would become ulcerated. Leishmania in the lesions were demonstrable by smear and culture. The serum immunoglobulins were normal except for an elevated IgM (IgG, 7.9 mg/ml: IgA. 4.2 mgiml; IgM, 4.9 mg/ml). C, level was normal ( 1.3 mg/ml). She was leishmanina-positive. MATERIALS
AND METHODS
Quantitative serum immunoglobulin assay was performed by the single radial diffusion technique of Mancini (17) using Behring standard serum and monospecific anti-human IgG. IgA, and IgM anti-rabbit sera (Behring, Frankfurt. Germany). Skin tests for delayed-type hypersensitivity (DTH) were performed with Lrishnrania tropica antigen (LTA). The antigen was prepared from Leishrmrnirr tropica grown on Novy-Nichol-McNeal medium with rabbit serum and overlaid with saline. Supernatant from the cultures containing leishmania was washed with buffered saline (pH 7.2 x 3) and preserved in phenol at a final concentration of 0.45%. Intradermal injection of 0.1 ml of 1: 10 and 1: 100 dilutions of the antigen gave a positive DRH reaction at 48 hr in individuals with a past history of cutaneous leishmania infection. Serum levels of leishmania-specific antibody were measured by the indirect fluorescent antibody technique (IFAT), using fluorescein-conjugated antiimmunoglobulin antisera (Behring). Transfer factor was prepared from healthy donors who had recovered from cutaneous leishmania infection in the past. Venous blood was collected in blood bags containing acid-citrate-dextrose and red cells were sedimented with dextran for 2 hr (Macrodex 6%, 10 ml/100 ml of blood). The leukocyte-rich supernatant was centrifuged at 2000 rpm for 15 min and the cells were washed and resuspended in buffered saline (pH 7.2) at a concentration of 1.0 x 10” cells/ml. The leukocyte suspension thus obtained was frozen and thawed 8 to 10 times, until no intact cells were seen microscopically, incubated with DNase at 37°C for 0.5 hr, and then vacuum-dialyzed at a pressure of 25 lb/cm”. The dialysate was filtered through a 0.22-nm Millipore filter. One milliliter of TFd, containing an amount approximately equivalent to the dialysate prepared from 5 x 10s cells, was frozen in vials at -70°C for later use. TFd was given by subcutaneous injection as near the skin lesion as possible. The dose and the frequency of TFd injections were monitored by the clinical response of the patient, as shown in Table 1.
TRANSFER
FACTOR
THERAPY
TABLE TRAMFER
FACTOR
THERAPY
Patient no. 1 1 1 2
x x x
1 ml/week, 4 months” 2 ml/week, 4 months 2 ml/week. 6 months
o 1 ml of TFd. TFd prepared from
185
OF LEISHMANIASIS
1
SCHEDULE
IN PATIENTS
WITH
CL1
Patient no. 2
Patient no. 3
1 x 1 ml/week, 2 months 1 x 2 ml/week, 6 months Nil. 1 month 2 x 2 ml/week, 5 months
1 x I ml/week. I month 1 x :! ml/week. 1 month 2 x 2 ml/week, 4 months 2 m1/4-6 weeks, 8 months
5.X
10” leukocytes.
RESULTS
The results of TFd therapy in the three patients with persistent CL1 are shown in Figs. 1 to 3. Complete healing of the lesions occurred in all three patients. Leg lesions in patient 1 (Fig. 1, A and B) began to show healing at 4 months and it took 14 months for the lesion to heal completely. In patient 2 (Fig. 2, C and D) clinical improvement of the facial lesions was noticed at 3 months. Following a gap in TFd therapy of 1 month, reactivation of the cutaneous lesions occurred at 8 months. Restitution of TFd therapy produced a prompt improvement in the lesion. The facial lesions healed completely by 14 months. A biopsy of the healed scar taken on completion of TFd therapy was negative for leishmania. In patient 3 (Fig. 3, E and F) facial lesions began to dry up by about 6 weeks. By 14 months there was complete clearing of the infected skin. Serum IgM in patients 1 and 3 returned to normal level (IgM, 1.5 and 1.2 mg/ml, respectively). Strongly positive DTH responses to LTA were observed in all three patients. All three patients remained free of infection 8 months after completion of TFd therapy. DISCUSSION
This apparently curative response to TFd therapy in three patients with persistent cutaneous leishmaniasis of several years duration, previously resistant to a variety of conventional therapeutic maneuvers, must clearly be viewed as very encouraging and worth vigorous supplementary studies. Although only three cases have been observed thus far healing was consistent and complete in each one in succession, and relapse and restoration to healing were produced concurrently with the withdrawal and restitution of TFd injections. It seems unlikely, therefore, that the cures can be attributed merely to chance. The mechanism of action of TFd itself is not known. It is believed that TFd induces an antigen-responsive state in lymphocytes so that they can subsequently undergo replication and proliferation in the presence of the appropriate antigen, in vitro and in vivo , and hence participate in cell-mediated immune responses ( 18). Persistence of infection occurred in the three patients with CL1 despite the development of DTH to leishmania antigens. It is therefore possible that TFd in these individuals caused elimination of the infective agent merely by increasing the number of antigen-responsive lymphocytes and, hence, a general augmentation of the host’s immune response. However, other possibilities for the mode of
186
SHARMA,
ANARAKI,
AND
ALA
TRANSFER
FACTOR
THERAPY
OF
LEISHMANIASIS
187
FIG. 1. Cutaneous leishmania lesion in patient 1 showing involvement of two-thirds of the left lower leg (A and Al). Following TFd, the cutaneous lesion healed completely. Only a deeply pigmented scar was left behind (B).
action of TFd therapy in these individuals also exist, such as selective effects on recruitment or function of T-cell subpopulations, or the capacity of the pooled preparations of TFd used, which covered a wide range of antigen specificities, to accommodate for any antigenic changes occurring in the parasite during its intracellular existence. The response to TFd therapy in patients with CL1 has been quite different from that in other intracellular infections. In the latter case, the infective agent was eliminated only partially or a relapse of the infection occurred, either during TFd therapy or on completion. The difference in results could be due to several factors. (i) TFd was injected locally. (ii) The total dose of TFd required for complete healing of the lesions was considerably larger than those used previously. (iii) TFd was prepared only from individuals with a past history of the infection, as opposed to skin test-positive individuals. (iv) The infective agent in CL1 is not ubiquitous so that the clinical course of the infection during TFd therapy was not complicated by reexposure to the infective agent.
188
SHARMA,
ANARAKI.
AND
AL.4
several isolated area of the face. There F IL. 2. Cutaneous leishmania lesions in patient 2 involved advancing edge to an otherwise dry lesion (Cl. After TFd therapy. the healing of the was an inflamed by absence of leishmania from the lesion on skin biopsy. (D). infl2 lmed edge was accompanied
FIG. 3. Cutaneous leishmania lesion in patient 3 involved the face as a single continuous lesion 03. the lesion near the angle of the mouth healed with mild scar formation, whe reas Follo\l ding TFd therapy skin around the scarred area was left behind in the malar region (F). a deep 11y pigmented
190
SHARMA,
ANARAKI,
AND
ALA
The results of TFd therapy in persistent CLI, therefore, warrant further double blind clinical studies on the effectiveness of TFd in intracellular infections. In the design of such studies, two important aspects of TFd therapy in intracellular infection will require serious consideration: First, intracellular infections in man generally tend to produce a spectrum of diseases and, second, the dose of TFd and its route of administration will need to be adjusted according to the target organ of infection. ACKNOWLEDGMENTS The authors wish to thank Dr. A. Nadim. patients included in the study. and R. Firooz, assistance.
the Dean of the School of Public Health, for referring the A. Baghian, and M. Zovein for their competent technical
REFERENCES I. Kurban, A. K.. Malak. J. A., and Farah, F. S.. Arch. Drrmatol. 93, 396, 1966. 2. Farah. F. A., and Malak. J. A.. Arch Dermatol. 103, 467, 1971. 3. Mauel, J.. Behin. R.. Noerjasin. B., and Holle, B., Presented at the Second Mononuclear Phagocyte Conference, Leiden, 1972. 4. Bray, R. S., Proc. Int. Congr. Parasitol. 3rd Munich A15, 257 (Abstract). 1974. 5. Nadim, A.. Proc. Int. Conpr. Parasitol. 3rd Munich AlS, 242 (Abstract), 1974. 6. Bullock, W. E.. Fields, J. P., and Brandiss. M. W.. N. Eng!. J. Med. 287, 1053, 1972. 7. Kirkpatrick. C. H.. Rich, R. R., and Smith, T. K., J. C/in. Invest. 51, 2948. 1972. 8. Kirkpatrick, C. H.. and Smith, T. K., J. Invest. Dermatol. 67, 425, 1976. 9. Schulkind. M. L., Adler 111, W. H., Altemeier 111, W. A., and Ayoub, E. M. 1 Cell. Imrnunol. 3, 606, 1972. IO. Valdimarson. H., Wood, C. B. S.. Hobbs, J. R.. and Holt, P. J. L., C/in. Erp. Immune/. 11, 151. 1972. 11. Valdimarsson, H..ln “Progress in Immunology” (L. Brent, and J. Holborow. Eds.). pp. 337-382. North-Holland. Amsterdam/American Elsevier. New York, 1974. 12. Catanzaro. A.. and Spitler, L., III “Transfer Factor” (M. S. Ascher, A. A. Gottlieb. and C. H. Kirkpatrick. Eds.), pp. 477-494, Academic Press, New York, 1975. 13. Graybill. J. R.. Ellenbogen. C., Drossman. D., Kaplan, P., and Thor, D. E., In “Transfer Factor” (M. S. Ascher. A. A. Gottlieb. and C. H. Kirkpatrick. Eds.) pp. 5099515, Academic Press, New York. 1975. 14. Heim. I,. R.. Bernhard, G.. Goldman. A. L., Dortf, G., and Rytel. M.. In “Transfer Factor” (M. S. Ascher. A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 457-464, Academic Press. New York, 1975. 15. Saha. H. K.. Mittal, M. M.. and Mahashwari. H. B., J. Clin. Microbial. 1. ??9. 1975. 16. Fudenberg, H. H.. Spider, L. E.. and Levin. A. S., Amer. .I. Pathol. 69, 529, 1972. 17. Mancini. G.. Carbonar. A. O., and Hermans, J. F., Immunochemistry 2, 235, 1965. 18. Lawrence. H. S.. Advan. Immunol. 11, 195, 1969.
,MM”NOLOGY
AND
IMMUNOPATHOLOGY
11,
183-190 (1978)
Preliminary Results of Transfer Factor Therapy Persistant Cutaneous Leishmania Infection MANJIT Iranian
K. SHARMA, National
FARVARDIN
Blood
Transfusion
ANARAKI, Service,
of
AND .F. ALA Tehran,
Iran
Received August 10, 1978 Three patients with persistent cutaneous leishmania infection of &, 14- and 30year duration, respectively, and with demonstrable delayed hypersensitivity to leishmania antigens were treated with leishmania-specific transfer factor prepared from healthy donors with a previous history of cutaneous leishmania infection. The cutaneous lesions healed completely in all three cases. The total dose of transfer factor required to achieve healing was much higher than the dose used by other investigators. The results justify a further clinical trial to determine the therapeutic effectiveness of transfer factor in cutaneous leishmaniasis. Further, because the infective agent is neither ubiquitous nor homogeneous, cutaneous leishmania infection in man offers a unique opportunity for a double blind study aimed at evaluating the therapeutic usefulness of transfer factor in human intracellular infections and further analytic studies to determine both the in vivo and in vitro specificity of transfer factor.
INTRODUCTION
Cutaneous leishmania infection (CLI) in man, as it is generally seen, is a selflimiting disease. The cutaneous lesion usually heals within months, although in rare cases, the cutaneous lesion persists for several years (1, 2). The causes of persistent lesions in an otherwise healthy individual are not known. Defects of the host’s immune response have been suggested as one possible explanation (1). Cell-mediated immunity (CMI) appears to play an important role in recovery from CLI, in both man and experimental animals. However, the development of CM1 is not always accompanied by recovery from the infection (3-5). Commonly used antiprotozoal drugs do not appear to change the course of self-limiting cutaneous lesions significantly and they are totally ineffective against persistent CLI. In the past, transfer factor (TFd), a dialyzable extract of leukocytes, has been used with some limited success in the treatment of a number of intracellular infections caused by mycobacteria, viruses, and fungi (6-16). These infections were associated with either normal or impaired CM1 to the infective agent in question. TFd therapy was attempted in three patients with persistent CL1 of several years duration who had failed to respond to antiprotozoal therapy in the past. CASE REPORT Patient I. A 35year-old
male presented with cutaneous leishmania infection of the left shin, persisting for the past 8 years. The skin lesion which was very small initially had progressed to involve most of the lower leg. The lesion was ulcerated and contained yellow cheesy material in several places. The skin adjoining the infected area showed minimal induration. His leishmanin skin test was positive, although 5 years after infection it was reported to be negative. His serum im183 0090-1229/‘79/020183-08$01.00/0 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved
184
SHARhl.4.
ASARAKI,
AND
:\I.:\
munoglobulins and C, levels were normal except for 1gM which was elevated (IgG, 14.2 mg/ml; IgA. 2.8 mg/ml; IgM. 3.4 mg/ml: C:,. 0.6 mgiml). Serum leishmania-specific fluorescent antibody titer was 1: 1024. Leishmania were demonstrable in lesions, by both smear and culture. PNtiettt _3. A 16-year-old male presented in whom cutaneous lesions of the face developed at the age of 2 years. The lesions appeared dry in the center and were surrounded by indurated margins. Leishmania in the infected area were demonstrable by skin biopsy. He was leishmanin-positive. His serum immunoglobulins were normal (IgG. 6.7 mg/ml: IgA. 3.2 mgiml: 1gM. 1.6 mgiml), and C., was low normal (0.5 mg/ml). Patient 3. A 33-year-old female presented in whom cutaneous lesions of the face developed at the age of 3 years. The lesions generally remained dry; however. from time to time they would become ulcerated. Leishmania in the lesions were demonstrable by smear and culture. The serum immunoglobulins were normal except for an elevated IgM (IgG, 7.9 mg/ml: IgA. 4.2 mgiml; IgM, 4.9 mg/ml). C, level was normal ( 1.3 mg/ml). She was leishmanina-positive. MATERIALS
AND METHODS
Quantitative serum immunoglobulin assay was performed by the single radial diffusion technique of Mancini (17) using Behring standard serum and monospecific anti-human IgG. IgA, and IgM anti-rabbit sera (Behring, Frankfurt. Germany). Skin tests for delayed-type hypersensitivity (DTH) were performed with Lrishnrania tropica antigen (LTA). The antigen was prepared from Leishrmrnirr tropica grown on Novy-Nichol-McNeal medium with rabbit serum and overlaid with saline. Supernatant from the cultures containing leishmania was washed with buffered saline (pH 7.2 x 3) and preserved in phenol at a final concentration of 0.45%. Intradermal injection of 0.1 ml of 1: 10 and 1: 100 dilutions of the antigen gave a positive DRH reaction at 48 hr in individuals with a past history of cutaneous leishmania infection. Serum levels of leishmania-specific antibody were measured by the indirect fluorescent antibody technique (IFAT), using fluorescein-conjugated antiimmunoglobulin antisera (Behring). Transfer factor was prepared from healthy donors who had recovered from cutaneous leishmania infection in the past. Venous blood was collected in blood bags containing acid-citrate-dextrose and red cells were sedimented with dextran for 2 hr (Macrodex 6%, 10 ml/100 ml of blood). The leukocyte-rich supernatant was centrifuged at 2000 rpm for 15 min and the cells were washed and resuspended in buffered saline (pH 7.2) at a concentration of 1.0 x 10” cells/ml. The leukocyte suspension thus obtained was frozen and thawed 8 to 10 times, until no intact cells were seen microscopically, incubated with DNase at 37°C for 0.5 hr, and then vacuum-dialyzed at a pressure of 25 lb/cm”. The dialysate was filtered through a 0.22-nm Millipore filter. One milliliter of TFd, containing an amount approximately equivalent to the dialysate prepared from 5 x 10s cells, was frozen in vials at -70°C for later use. TFd was given by subcutaneous injection as near the skin lesion as possible. The dose and the frequency of TFd injections were monitored by the clinical response of the patient, as shown in Table 1.
TRANSFER
FACTOR
THERAPY
TABLE TRAMFER
FACTOR
THERAPY
Patient no. 1 1 1 2
x x x
1 ml/week, 4 months” 2 ml/week, 4 months 2 ml/week. 6 months
o 1 ml of TFd. TFd prepared from
185
OF LEISHMANIASIS
1
SCHEDULE
IN PATIENTS
WITH
CL1
Patient no. 2
Patient no. 3
1 x 1 ml/week, 2 months 1 x 2 ml/week, 6 months Nil. 1 month 2 x 2 ml/week, 5 months
1 x I ml/week. I month 1 x :! ml/week. 1 month 2 x 2 ml/week, 4 months 2 m1/4-6 weeks, 8 months
5.X
10” leukocytes.
RESULTS
The results of TFd therapy in the three patients with persistent CL1 are shown in Figs. 1 to 3. Complete healing of the lesions occurred in all three patients. Leg lesions in patient 1 (Fig. 1, A and B) began to show healing at 4 months and it took 14 months for the lesion to heal completely. In patient 2 (Fig. 2, C and D) clinical improvement of the facial lesions was noticed at 3 months. Following a gap in TFd therapy of 1 month, reactivation of the cutaneous lesions occurred at 8 months. Restitution of TFd therapy produced a prompt improvement in the lesion. The facial lesions healed completely by 14 months. A biopsy of the healed scar taken on completion of TFd therapy was negative for leishmania. In patient 3 (Fig. 3, E and F) facial lesions began to dry up by about 6 weeks. By 14 months there was complete clearing of the infected skin. Serum IgM in patients 1 and 3 returned to normal level (IgM, 1.5 and 1.2 mg/ml, respectively). Strongly positive DTH responses to LTA were observed in all three patients. All three patients remained free of infection 8 months after completion of TFd therapy. DISCUSSION
This apparently curative response to TFd therapy in three patients with persistent cutaneous leishmaniasis of several years duration, previously resistant to a variety of conventional therapeutic maneuvers, must clearly be viewed as very encouraging and worth vigorous supplementary studies. Although only three cases have been observed thus far healing was consistent and complete in each one in succession, and relapse and restoration to healing were produced concurrently with the withdrawal and restitution of TFd injections. It seems unlikely, therefore, that the cures can be attributed merely to chance. The mechanism of action of TFd itself is not known. It is believed that TFd induces an antigen-responsive state in lymphocytes so that they can subsequently undergo replication and proliferation in the presence of the appropriate antigen, in vitro and in vivo , and hence participate in cell-mediated immune responses ( 18). Persistence of infection occurred in the three patients with CL1 despite the development of DTH to leishmania antigens. It is therefore possible that TFd in these individuals caused elimination of the infective agent merely by increasing the number of antigen-responsive lymphocytes and, hence, a general augmentation of the host’s immune response. However, other possibilities for the mode of
186
SHARMA,
ANARAKI,
AND
ALA
TRANSFER
FACTOR
THERAPY
OF
LEISHMANIASIS
187
FIG. 1. Cutaneous leishmania lesion in patient 1 showing involvement of two-thirds of the left lower leg (A and Al). Following TFd, the cutaneous lesion healed completely. Only a deeply pigmented scar was left behind (B).
action of TFd therapy in these individuals also exist, such as selective effects on recruitment or function of T-cell subpopulations, or the capacity of the pooled preparations of TFd used, which covered a wide range of antigen specificities, to accommodate for any antigenic changes occurring in the parasite during its intracellular existence. The response to TFd therapy in patients with CL1 has been quite different from that in other intracellular infections. In the latter case, the infective agent was eliminated only partially or a relapse of the infection occurred, either during TFd therapy or on completion. The difference in results could be due to several factors. (i) TFd was injected locally. (ii) The total dose of TFd required for complete healing of the lesions was considerably larger than those used previously. (iii) TFd was prepared only from individuals with a past history of the infection, as opposed to skin test-positive individuals. (iv) The infective agent in CL1 is not ubiquitous so that the clinical course of the infection during TFd therapy was not complicated by reexposure to the infective agent.
188
SHARMA,
ANARAKI.
AND
AL.4
several isolated area of the face. There F IL. 2. Cutaneous leishmania lesions in patient 2 involved advancing edge to an otherwise dry lesion (Cl. After TFd therapy. the healing of the was an inflamed by absence of leishmania from the lesion on skin biopsy. (D). infl2 lmed edge was accompanied
FIG. 3. Cutaneous leishmania lesion in patient 3 involved the face as a single continuous lesion 03. the lesion near the angle of the mouth healed with mild scar formation, whe reas Follo\l ding TFd therapy skin around the scarred area was left behind in the malar region (F). a deep 11y pigmented
190
SHARMA,
ANARAKI,
AND
ALA
The results of TFd therapy in persistent CLI, therefore, warrant further double blind clinical studies on the effectiveness of TFd in intracellular infections. In the design of such studies, two important aspects of TFd therapy in intracellular infection will require serious consideration: First, intracellular infections in man generally tend to produce a spectrum of diseases and, second, the dose of TFd and its route of administration will need to be adjusted according to the target organ of infection. ACKNOWLEDGMENTS The authors wish to thank Dr. A. Nadim. patients included in the study. and R. Firooz, assistance.
the Dean of the School of Public Health, for referring the A. Baghian, and M. Zovein for their competent technical
REFERENCES I. Kurban, A. K.. Malak. J. A., and Farah, F. S.. Arch. Drrmatol. 93, 396, 1966. 2. Farah. F. A., and Malak. J. A.. Arch Dermatol. 103, 467, 1971. 3. Mauel, J.. Behin. R.. Noerjasin. B., and Holle, B., Presented at the Second Mononuclear Phagocyte Conference, Leiden, 1972. 4. Bray, R. S., Proc. Int. Congr. Parasitol. 3rd Munich A15, 257 (Abstract). 1974. 5. Nadim, A.. Proc. Int. Conpr. Parasitol. 3rd Munich AlS, 242 (Abstract), 1974. 6. Bullock, W. E.. Fields, J. P., and Brandiss. M. W.. N. Eng!. J. Med. 287, 1053, 1972. 7. Kirkpatrick. C. H.. Rich, R. R., and Smith, T. K., J. C/in. Invest. 51, 2948. 1972. 8. Kirkpatrick, C. H.. and Smith, T. K., J. Invest. Dermatol. 67, 425, 1976. 9. Schulkind. M. L., Adler 111, W. H., Altemeier 111, W. A., and Ayoub, E. M. 1 Cell. Imrnunol. 3, 606, 1972. IO. Valdimarson. H., Wood, C. B. S.. Hobbs, J. R.. and Holt, P. J. L., C/in. Erp. Immune/. 11, 151. 1972. 11. Valdimarsson, H..ln “Progress in Immunology” (L. Brent, and J. Holborow. Eds.). pp. 337-382. North-Holland. Amsterdam/American Elsevier. New York, 1974. 12. Catanzaro. A.. and Spitler, L., III “Transfer Factor” (M. S. Ascher, A. A. Gottlieb. and C. H. Kirkpatrick. Eds.), pp. 477-494, Academic Press, New York, 1975. 13. Graybill. J. R.. Ellenbogen. C., Drossman. D., Kaplan, P., and Thor, D. E., In “Transfer Factor” (M. S. Ascher. A. A. Gottlieb. and C. H. Kirkpatrick. Eds.) pp. 5099515, Academic Press, New York. 1975. 14. Heim. I,. R.. Bernhard, G.. Goldman. A. L., Dortf, G., and Rytel. M.. In “Transfer Factor” (M. S. Ascher. A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 457-464, Academic Press. New York, 1975. 15. Saha. H. K.. Mittal, M. M.. and Mahashwari. H. B., J. Clin. Microbial. 1. ??9. 1975. 16. Fudenberg, H. H.. Spider, L. E.. and Levin. A. S., Amer. .I. Pathol. 69, 529, 1972. 17. Mancini. G.. Carbonar. A. O., and Hermans, J. F., Immunochemistry 2, 235, 1965. 18. Lawrence. H. S.. Advan. Immunol. 11, 195, 1969.
