Chronic Mucocutaneous Candidiasis: Model-Building in Cellular Immunity

Transcription

1 NIH Conference Chronic Mucocutaneous Candidiasis: Model-Building in Cellular Immunity Moderator: CHARLES H. KIRKPATRICK, M.D. Discussants: ROBERT R. RICH, M.D., and JOHN E. BENNETT, M.D., Bethesda, Maryland Infection with Candida albicans develops in diverse clinical settings. It may be minimal or life-threatening in severity, depending on the site of infection and clinical circumstance. Chronic mucocutaneous candidiasis is an uncommon superficial infection of the skin, nails, and oral and vaginal mucous membranes, and is often associated with defects in cellular immune function. Untreated, the course is usually unremitting. Intravenous amphotericin B often results in regression or clearing of infection, but relapse generally occurs after discontinuation of therapy. Twelve patients with chronic mucocutaneous candidiasis have been studied at the National Institutes of Health. Specific defects in cellular immunity have been characterized in eight of these patients. Cutaneous anergy to Candida antigens has been associated with defective lymphocyte function in vitro. Temporary modification of cellular immune function in one case was associated with partial clearing of skin lesions. Experience with reconstitution of defects in cellular immunity to date is reviewed here to provide guidelines for future efforts. * Head, Section of Clinical Allergy and Hypersensitivity, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases. An edited transcription of a Combined Clinical Staff Conference at the Clinical Center, Bethesda, Md., by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health, Education, and Welfare. DR. CHARLES H. KIRKPATRICK* : Delayed (cellular) hypersensitivity is a consequence of natural exposure to certain bacteria and fungi. The role of this response in resistance to infection with these organisms is uncertain. Candidiasis, however, regularly complicates the clinical courses of patients with severe defects in cellular immunity. Immune responses in patients with mucocutaneous candidiasis have been studied in an attempt to identify defects that predispose them to chronic infection with Candida species. Definition of host-related abnormalities serves to identify factors that maintain the commensal relationship between the host and potential program and might permit development of therapeutic programs directed at reconstitution of the deficient host. Candidiasis Syndromes Dr. Robert R. Richf: Candida infection has become a problem of substantial interest within the broad context of questions about human cellular immunity. We have directed our attention primarily to a specific clinical entity, chronic mucocutaneous candidiasis. Before focusing on this disease, however, certain general comments regarding the biology of the organism, its relationship to the host, and the clinical spectrum of Candida infections are necessary. Candida albicans, the commonest but not invariable species involved in human infection, is found throughout the world. It exists primarily in a commensal relationship with man but has also been isolated from a large variety of birds and animals (1). It is a dimorphic fungus, exhibiting both yeast and filamentous growth. It is cultured most frequently from the human gastrointestinal tract. In a t Senior Staff Fellow, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases. Annals of Internal Medicine 74: ,

2 recent study of normal adults, Candida albicans was recovered from 30% of oropharyngeal cultures, and 54% of jejunal, 55% of ileal, and 65% of fecal samples (2). On the other hand, the fungus is infrequently found in the microflora of normal skin (3) and is rarely cultured from air (4) or from soil uncontaminated with feces (5). The organism will grow on a wide variety of laboratory media. It will attack keratin in vitro and can use it as a sole source of nitrogen (6). HOST-ORGANISM RELATIONSHIP In any study of disease caused by an organism that usually enjoys a commensal relationship with its host, understanding of the pathogenesis commonly derives from elucidation of host impairments. These deficiencies favor a shift from a stable host-parasite relationship to parasitic invasion and proliferation. Such defects may be multiple and are the hallmark of opportunistic infection. Candidiasis ranges from mild self-limited superficial infections to overwhelming systemic disease of extremely poor prognosis. The extent to which host defenses are compromised is, in general terms, proportional to the seriousness of the infection. Characterization of specific problems in the cellular immune mechanisms of patients with chronic mucocutaneous candidiasis has been a major aim of our investigations. But certain nonimmunologic factors that favor Candida growth or invasion can also be identified. The first of these is physical disruption of the integument. It is usually attributable to tissue maceration or trauma and is often associated with the commonest form of candidiasis, acute self-limited skin or mucous membrane infection. It often appears to be of fundamental pathogenetic importance in Candida intertriginous infection (7) or paronychia (8). Its effect has also been demonstrated in experimental Candida infections. Candidiasis is not established by inoculation of yeast into a normal mouth, but mild abrasion of the oral mucous membranes, followed by Candida inoculation, may result in clinical infection (9). Also, gentle scraping of a nail fold, followed by occlusion for 1 to 3 weeks, will frequently produce positive Candida cultures, although tissue infection has not been achieved in this manner (8). Certain local and systemic growth factors have been associated with Candida infection. The increased frequency of isolated vaginal candidiasis in pregnancy (10) and diabetes mellitus (9) has prompted study of the vaginal microenvironment in these conditions. Although data remain inferential, glycogen content of vaginal epithelium is increased in these states, and fermentation by lactobacilli presumably lowers vaginal ph to the 5 to 6 range optimal for yeast phase growth (9). Experimental vaginal candidiasis in mice, however, does not correlate with glycogen content (11), and other explanations may eventually prove to be correct. Iatrogenic factors may also unbalance a hostparasite relationship. The effect of antibiotics, particularly wide-spectrum antibiotics, and corticoid therapy on emergence or exacerbation of Candida infection has been widely discussed. The mechanisms of growth promotion in these circumstances are not entirely clear, but it seems probable that antibiotics enhance Candida overgrowth primarily by suppression of bacterial competitors for nutritive factors (9). There is little evidence to support alternative explanations, such as a direct stimulatory effect of antibiotics on Candida growth or virulence. Although the effect of corticoids is also uncertain, these agents appear to enhance invasiveness rather than replication, presumably by further compromising host defense mechanisms (12, 13). The contribution of multiple host factors to the development of candidiasis is particularly apparent in certain states associated with marked debility or severe compromise of normal defenses. Severe malnutrition or malabsorption, serious blood dyscrasias, particularly leukemia and lymphomas, advanced malignancies, and the postoperative state most commonly underlie serious Candida infections (9). Both endogenous and iatrogenic factors are often identified in these cases. Furthermore, patients with chronic mucocutaneous candidiasis often relate the initial appearance of lesions to a course of antibiotics, and many report exacerbation of candidiasis when antibiotics are required after establishment of the disease. SPECTRUM OF CANDIDIASIS Having identified certain nonimmunologic host factors that apparently predispose to candidiasis, we will briefly recapitulate the clinical spectrum of the disease. It is important to recognize that, although chronic mucocutaneous infection is best suited to a study of specific defects in human cellular immunity, it represents but a single presentation of clinical candidiasis. Clearly most common are the acute, superficial, and usually self-limited infections of oral and vaginal mucous membranes. These are seen most often in denture wearers (14), infancy, pregnancy, and diabetes (9). Isolated Candida vaginitis is often encountered as a minor infection of adult women who otherwise appear physiologically normal. The frequency of oral and vaginal infection suggests that these are the sites in which man's commensal 956 June 1971 Annals of Internal Medicine Volume 74 Numbers

3 relationship with the organism is most tenuous and easily threatened. Paronychial infection is likewise often an isolated jinding. A history of tissue trauma or maceration is often obtained. Circulatory impairment may also be present. Nail infection is characteristically chronic and resistant to therapy; healing depends on clearing of nail-fold infection. It is often self-limited, running a course of months to years (8). A second category of candidiasis is systemic infection, either of the blood stream or visceral organs. Candida sepsis may result from seeding by an infected prosthetic device in the blood stream, for example, an indwelling intravenous catheter or shunt. Simple removal of the infected device is often curative in these cases (15). Candida is a rare cause of vegetative endocarditis; it is encountered particularly with the endocarditis of narcotics addiction (16). Successful treatment of these patients is rare. Prognosis is also unfavorable in Candida sepsis or disseminated visceral infection that develops in a setting of severe general debility or malignancy. Gastrointestinal, pulmonary, and renal infections are most commonly encountered, although any organ may be involved (9). It should be emphasized that simply culturing Candida from urine or sputum is not sufficient to establish a diagnosis of tissue infection. Systemic therapy should be initiated only when other clinical features also suggest a diagnosis of visceral candidiasis. An important but often neglected diagnostic aid in this regard is the direct microscopic examination of tissue or material obtained for culture. Although debate continues regarding the role of mycelial versus yeast growth phases in establishment of tissue infection, there is' little doubt that filamentous forms Figure 1. Extensive chronic candidiasis of the nails and skin of the hands and distal forearms. are almost always concomitants of clinically significant infection (17). The failure to demonstrate mycelia in freshly prepared smears is evidence against the diagnosis. Chronic Mucocutaneous Candidiasis This relatively uncommon variety of candidiasis is characterized by development, usually in infancy or childhood, of chronic, often widespread, infection of skin, nails, and mucous membranes. Involvement may extend from the oropharynx to the esophagus, but further visceral extension or sepsis is practically unknown. Oral thrush and vaginitis closely resemble the acute infections previously described, except for their remarkable chronicity. Cutaneous disease tends to be of acral distribution Figure 2A. Candidiasis of the thumbs producing nail destruction and cutaneous horn formation. B. Similar cutaneous changes secondary to chronic candidiasis of the feet. Kirkpatrick et al. Chronic Mucocutaneous Candidiasis 957

4 margin of involvement is sharply demarcated, and adjacent uninfected skin appears entirely normal. Except for perineal infections and candidiasis of infancy, the trunk is generally free of disease. Hyperkeratosis and parakeratosis of the epidermis are seen microscopically (Figure AA). The dermis is often intensely infiltrated with chronic inflammatory cells, primarily lymphocytes and plasma cells. Multinucleated giant cells may also be encountered. Methenamine silver stain shows the fungus residing within the keratinized layer. The superficial nature of the infection can be appreciated in Figure 4B. Fungal invasion, even of underlying dermis or hair shafts, is distinctly unusual. The dimorphic character of the organism in clinical infections is seen at higher magnification; both yeast and mycelial forms are easily found. Figure 3. Serpiginous appearance of chronic candidiasis of the skin of the popliteal fossa. Note the sharp demarcation between affected and normal skin. (Figure 1). Lower extremities are infected somewhat less frequently than hands and arms. Lesions are red, raised, serpiginous, and scaling, often with marked hyperkeratosis. In contrast with oral or vaginal disease, cutaneous lesions are not tender or painful. Severe dystrophic changes of nails are usually seen. Chronic swelling of the nail fold and marked thickening, distortion, and fragmentation of the nail substance are apparent. The severely infected thumbs of one patient and the feet of another are demonstrated in Figure 2. Striking hyperkeratosis with cutaneous horn formation are predominant features of the disease in these patients. They are the clinical features of a subtype designated Candida granuloma (18). The scalp is also often infected, and thick, plaque-like hyperkeratotic scales may be seen. The proximal extremities are less commonly involved than the hands and feet; disease here tends to be less severe, and there is less hyperkeratosis. Proximal lesions tend to be less confluent, and their serpiginous character may be more obvious. The typical geographic nature of the infection is well illustrated in Figure 3. The ASSOCIATED DISORDERS Chronic mucocutaneous candidiasis is recognized in several clinical settings. First are the diseases associated with morphologic abnormalities of the thymus and thymus-dependent tissues. The immunologic defects in these patients may be primarily restricted to profound deficiencies of the cellular immune system, for example, the DiGeorge (19, 20) and Nezelof-Allibone (21) syndromes. Others of this group have combined deficiencies, with severe defects of both cellular and humoral immunity. These include patients with Swiss-type agammaglobulinemia and thymus dysplasia (22). Mucocutaneous candidiasis is an almost invariable feature of these diseases. The prognosis, particularly of patients with the combined defects, is extremely poor. Death usually occurs before 2 years of age. Although it is the most consistent infectious problem in these patients, candidiasis does not commonly determine the outcome. Death is most frequently the result of bacterial or viral pneumonia; even in these severely compromised patients tissue invasion or dissemination of Candida is unusual. Chronic candidiasis is also seen in patients without lethal immunologic deficiencies; all of our patients belong to this latter group. A traditional differentiation is made between those with and without clinically significant endocrine disease. Since candidiasis usually develops before endocrinopathy, however, this differentiation is not often helpful in classification of newly diagnosed cases. Our experience in study of these patients has produced a generally characteristic clinical series (Table 1). Both males and females are affected. Onset of candidiasis is usually in early childhood, with a mean age of 3 years for our patients. Most patients were studied in adolescence, with an age range of 6 to 26 years at the 958 June 1971 Annals of Internal Medicine Volume 74 Number 6

5 Table 1. Clinical Presentation of Patients with Chronic Mucocutaneous Candidiasis Sites Affected Patient Sex Onset of Age Candidiasis Studied Mouth Nails Skin Vagina < yr y S.H. M 1/12 10 J.C. F 1/ D.L. M 6/12 20 M.L. M 8/12 6 R.H. M 9/ G.R. M 2 24 R.M. M 2 16 W.M. M S.M. F H.S. M 6 9 J.B. F 8 23 K.B. F Mean 3 17 time of investigation. All patients in our series had infection of mouth and nails, all females had vaginal candidiasis, and 10 of the 12 patients also had skin involvement. Seven of our patients related a history of other significant infectious diseases (Table 2), and these were most frequently pyogenic. Two of the 12 patients had a second fungal disease, in both cases a chronic dermatophyte infection. From one patient Trichophyton tonsurans was cultured. The second patient had an impressive exophytic dermatophyte infection of the foot and leg (Figure 5). Epidermophyton floccosum, but not Candida, was recovered from the lesion. Griseofulvin and tolnaftate therapy resulted in dramatic improvement. The importance of physical disruption of the integument to development of self-limited candidiasis, and the apparent predisposition of patients with chronic mucocutaneous disease to other cutaneous infections, lead one to examine these patients for congenital or acquired ectodermal defects. The uninfected skin of these patients usually appears normal, although dystrophic changes in skin and hair have been reported (23). One structural problem in ectoderm development is readily apparent, however, on dental examination (Figure 6). Teeth are usually dysplastic, and some may be congenitally missing. Severe malocclusion is often present, and enamel formation is severely defective. Eleven of our patients had significant dental dysplasia; three had dentures when first seen. Malabsorption syndromes, keratoconjunctivitis, and chronic hepatitis have also been reported in other series of chronic candidiasis patients (23, 24). Three of our patients (R.M., W.M., M.L.) had steatorrhea, two (R.H. and G.R.) had chronic keratoconjunctivitis, and two (D.L. and G.R.) had chronic hepatitis. Australia antigen was detected by immunodiffusion in the serum of both of the latter cases. Congenital cystic disease of the lung was seen in one patient (D.L.), with severe recurrent pyogenic infections. As previously noted, endocrine failure is a frequent concomitant of the mucocutaneous candidiasis syndrome (Table 3). Mean age of onset of endocrine disease was 12.8 years in our patients, considerably later than the mean age of 3 years for development Figure 4A. Histologic appearance of chronic cutaneous candidiasis of the calf. Note the intense inflammatory infiltrate in the dermis. Hyperkeratosis and parakeratosis were present throughout the epidermis. (Hematoxylin and eosin; original magnification, X 100.) B. Methenamine-silver stain of skin biopsy. Note that the fungi are limited to the keratinized layer. (Original magnification, X 100.) Kirkpatrick et al. Chronic Mucocutaneous Candidiasis 959

6 Figure 5. Exophytic dermatitis caused by Epldermophyton f/occosum in a patient with chronic candidiasis. of fungal disease. In none of these cases was endocrine disease clinically apparent before the candidiasis, although this is not invariably the case in published reports (25). Five patients had endocrinopathy. Hypoparathyroidism, the most frequently associated endocrine disease in the literature (24, 25), was diagnosed only once. Parathyroid disease may, of course, be associated with candidiasis by two pathogenetically distinct means. First is the third and fourth pharyngeal pouch syndrome of DiGeorge. These patients fail to develop both thymus and parathyroid glands from the embryonic anlage (26). Soon after birth they present with hypocalcemic tetany. It is conceivable that a group of patients with an incomplete and less severe form of this disease will eventually be recognized; if so, our patient (R.H.), who also has a defect in thymus-dependent immune function (27), may represent such an example. A more probable explanation, however, is that our patient's parathyroid glands were involved after 7 years of age in a poorly understood dystropic or abiotropic process that also rendered three of our patients hypoadrenal, two hypothyroid, and one diabetic. Whatever the pathogenetic mechanism involved, it seems clear that the associated endocrine disease is not a direct effect of Candida infection. Candida has never been cultured from an affected endocrine gland, and no evidence has been found for Candida dissemination in those cases that have been autopsied (24). Moreover, kindreds have been identified in which siblings of a propositus with candidiasis had hypoparathyroidism or Addison's disease without evidence of Candida infection (23, 24, 28). Tissue antibodies have been frequently detected in these patients. Seven of our 12 patients had at least one autoantibody (Table 3). Antinuclear antibodies were never detected, and lupus erythematosus preparations were consistently negative. Of course, the detection of autoantibodies cannot be regarded as evidence for an immunologically mediated pathogenesis for the endocrinopathies in these patients. While this is an attractive hypothesis in patients with defective cellular immunity, there is no clear evidence that the antibodies are pathogenic. They may only be concomitants of the disease or "markers" indicative of genetic predisposition to immunologic diseases (23). GENETIC FACTORS Genetic determinants may have an important role in the development of at least some cases of chronic candidiasis. Although sporadic cases are the most frequently encountered, the syndrome of candidiasis with endocrine disease is clearly familial in some instances. Two of the patients in this series are brothers (R.M. and W.M.). One developed chronic mucocutaneous candidiasis at 2 years of age, the other at 3 years of age. Addison's disease appeared abruptly in both at 8 years of age (29). Another of Table 2. Noncandidal Infections in Patients With Mucocutaneous Candidiasis Patient Bacterial Infections Viral Infections Fungal Infections SIL Staphylococcal pyoderma Trichophyton tonsurans J.C. Staphylococcal pyoderma Recurrent herpes zoster Epidermophyton floccosun D.L. Staphylococcal pyoderma Recurrent pneumonia M.L. R.H. Pneumococcal sepsis Chronic otitis media G.R. Pneumonia Recurrent respiratory Septic arthritis infections R.M. W.M. S.M. H.S. Recurrent pneumonia Recurrent pneumonia J.B. Staphylococcal pyoderma K.B. Pneumonia 960 June 1971 * Annals of Internal Medicine Volume 74 Number 6

7 our patients (G.R.) has been the subject of an extensive family survey elsewhere (23). Although only the propositus had either candidiasis or endocrine disease, 17 of 28 relatives had one or more serum autoantibodies, including rheumatoid factor and adrenal, thyroid, uvea, colon, and thyroglobulin antibodies. Examination of available kindreds (23-25, 28-30) shows horizontal rather than vertical inheritance of candidiasis associated with endocrine disease, suggesting autosomal recessive transmission of the predisposition (31). One of our patients (S.H.) also had a family history remarkable for its prevalence of the ectodermal diseases most frequently associated with candidiasis (Figure 7). Ten of the 18 family members of the patient's generation from whom information was available had severe dental dysplasia, requiring fullmouth extractions and dentures by 20 years of age. Five of 10 members of the father's generation, as well as the patient, had a history of severe chronic furunculosis. NATURAL HISTORY To conclude this survey of candidal syndromes we will briefly examine the natural history of chronic candidiasis. The prognosis in patients with profound morphologic abnormalities of the thymus is extremely poor. With traditional therapy, mortality has been 100%, with most patients succumbing to bacterial or viral infection. Among patients with candidiasis and endocrine failure, the outlook has also been relatively unfavorable. Of 37 patients with these diseases in one review, 14 died between the ages of 4 and 16 Figure 6. Severe dental dysplasia in a 12-year-old patient with chronic candidiasis. Note thrush of the mucous membranes, pyorrhea, and carious teeth, and dysplastic enamel. Table 3. Endocrine Diseases and Autoantibodies in Patients with Mucocutaneous Candidiasis Patient Endocrine Disease Autoantibodies Onset of Endocrinopathy yr S.H. J.C. None Hypothyroidism Rheumatoid factor None 21 D.L. None None M.L. None Antiadrenal Antithyroid Rheumatoid factor R.H. Hypoparathyroidism Antithyroglobulin 7 G.R. Addison's disease Hypothyroidism Antiadrenal Antithyroid 20 Diabetes mellitus Antithyroglobulin Rheumatoid factor R.M. Addison's disease None 8 W.M. Addison's disease Antithyroglobulin 8 S.M. None None H.S. None None J.B. None Antithyroid Rheumatoid factor K.B. None Rheumatoid factor years (25). In our group of twelve patients, there has been one death (G.R.) at 24 years of age, probably in addisonian crisis. Two features of this disease must be emphasized. First, the endocrinopathy may present in an explosive and life-threatening manner (29), and this should always be considered in an acutely ill patient with candidiasis. Second, progressive polyendocrinopathy is not unusual, and all patients with chronic candidiasis deserve periodic complete endocrine reevaluations. The natural history of mucocutaneous candidiasis without endocrinopathy is unclear. The course is certainly chronic, in terms of decades. Nonetheless, the substantial majority of these patients are less than 30 years of age. It seems unlikely that all these patients developed endocrine failure and succumbed with this complication before 30 years of age. It appears equally improbable that a large number of older patients with a disease as visually dramatic as this should not have come to medical attention. One is consequently tempted to postulate an eventual spontaneous remission for at least some of these patients. Unfortunately, this has not as yet been substantiated, and only very long periods of careful clinical study will eventually elucidate the natural history of this disease in its uncomplicated form. Chemotherapy of Chronic Candidiasis Dr. John E. Bennett*: Chemotherapy of chronic mucocutaneous candidiasis has left much to be desired, underscoring the potential value of immuno- * Head, Infectious Disease Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases. Klrkpatrlck et at. Chronic Mucocutaneous Candidiasis 961

8 logic reconstitution. The only treatment leading consistently to improvement has been amphotericin B, given intravenously. The published experience with this agent will be reviewed briefly. In order to select patients comparable to those discussed by Drs. Kirkpatrick and Rich, patients were included only if they met all the following criteria: [1] chronic oral thrush with characteristic hypertrophic skin lesions (Figures 1 to 3), one or both sites yielding Candida albicans on culture; [2] onset before 20 years of age; and [3] duration of intravenous amphotericin B therapy exceeding 1 week. Patients with thrush and skin infection involving only nails, paronychia, or mucocutaneous junction were not included. Thirtytwo published cases met these criteria (32-66). Five cases were excluded from further analysis because data about treatment and results were insufficient (62-66). For those interested in studying the original publications about the other 27 cases, the following should be noted. Five patients are cited twice (41-42, 45-46, 47-48, 56-57, 58-59). In addition, one publication (37) not only reports a case but also gives further information about three previously published cases (36, 58, 60). Two reports (33, 56) each include two cases meeting the above criteria. Of the 27 patients, an important additional measure of confidence in the diagnosis was given by demonstration of pseudohypae in smear or biopsy from mucocutaneous lesions of all but 1 patient (35). Location of the typical hyperkeratotic, often crusted, lesion included the face or scalp in all but one patient, in whom the skin of the trunk and extremities was involved (49). In all but two cases (37, 43) the nails were mentioned as infected. AMPHOTERICIN B All 27 patients were considered to be improved as a result of intravenous amphotericin B therapy. Although glabrous skin usually cleared more rapidly, improvement in scalp lesions also occurred. Face, scalp, and trunk lesions specifically were stated to clear completely in 18 cases. Thrush disappeared in 15 cases. Nail infections often improved slightly, but, unless the nail was removed, total regrowth of completely normal nail by time of reporting was not specifically mentioned by any author. Complete and lasting eradication of all clinical evidence of disease proved to be an elusive goal. Only one patient was said to be clearly free of all manifestations of disease for more than 12 months (44). This patient, a 13-year-old boy, was given a total dose of 1,000 mg in one course because of concomitant cryptococcal meningitis. One and one half years later he was free of all signs of candidiasis. The nails were said to be dystropic but free of fungi on potassium hydroxide smear. Eight other patients were said to be free of lesions at time of reporting (32, 38, 39, 42, 48, 51, 54, 55). Only one of these clearly received less than 6 weeks of intravenous amphotericin B (39). Dermabrasion of face lesions perhaps expedited the response in this patient, as well as in another patient (38) among this group of eight. A variety of other therapeutic measures was used in most of the 27 patients during and after intravenous amphotericin B in hopes of facilitating or maintaining remission. These included topical keratolytic agents, as well as topical and oral polyene antibiotics. These measures generally had proved to be useless before Figure 7. Pedigree of Patient S.H., showing the frequent occurrence of dental dysplasia and furunculosis among paternal relatives. The index case is indicated by the arrow. 962 June 2971 Annals of Internal Medicine Volume 74 Number 6

9 parenteral therapy and were not credited with much effect later. The experience of Drs. Kirkpatrick and Rich at this institution was in keeping with the published reports. They have treated two such patients with intravenous amphotericin B, each receiving a total dose of 1,000 mg over 6 weeks. Both patients had total clearing of skin and mouth lesions. Infected nails were avulsed. One patient is currently under bimonthly therapy and in remission. The other has not returned for evaluation after treatment. It is clear that amphotericin B has shown a limited ability to eradicate chronic mucocutaneous candidiasis permanently. This should not obscure the fact that cosmetically deforming, sometimes secondarily infected face and scalp lesions have cleared the most readily and relapsed the least frequently. These sites were still free of lesions at time of reporting in 17 of 27 patients. In contrast, thrush often relapsed and remained the patient's most symptomatic lesion and was absent at time of reporting in only nine patients. The authors believe that this amelioration warrants the considerable toxicity and expense of intravenous amphotericin B. OTHER THERAPIES Experience with forms of therapy other than intravenous amphotericin B has yielded only an occasional definite chemotherapeutic response. In contrast to the usual poor response to oral amphotericin B, one patient seemed to absorb enough of this insoluble drug to show marked improvement (67). A lesser but still definite degree of improvement in one patient was noted with topical amphotericin B dissolved in dimethylsulfoxide (44). A combination of topical measures, including gentian violet, has occasionally met some enthusiasm (68). Removal of infected nails often has been performed, but regrowth of infected nail has seemed assured unless candidiasis had been controlled elsewhere. Two new oral antifungal agents are being evaluated in candidiasis: 5-fluorocytosine (69) and clotrimazole (70). Neither drug has been evaluated enough in the therapy of chronic mucocutaneous candidiasis to warrant an opinion as to its merit. The Deficient Host Dr. Kirkpatrick: Immunological competence requires that an organism have the capacity to recognize antigenic substances as foreign, be able to modify or "process" antigens so they interact with antigen-sensitive cells, and finally be able to differentiate cells that produce the appropriate effectors such as specific antibodies or mediators of cellular immunity. Defects at any stage in the sequence may impair the final response and render the individual susceptible to infectious diseases. Many of the crucial observations in developmental immunology have occurred during the past 10 years, and it is appropriate to review briefly some of the current concepts of differentiation and division of labor in the immune response. In the early 1960's it was reported that thymectomy of newborn mice markedly reduced their capacity to mount an immune response (71). The cell-mediated responses such as delayed hypersensitivity, graft-versus-host reactions, and allograft rejection were most severely affected: the effect on antibody production was somewhat variable, depending on the antigen and the animals used. These observations implied that cellular and humoral responses differed in their dependence on thymus function and suggested that the immune system may have two more or less independent components. This hypothesis has received support both from studies of the immune system in chickens (72) and from observations on individuals with immunologic deficiency syndromes (73). The Two-Component Concept of the Immune Response Chickens have three major lymphoid organs: the thymus, the bursa of Fabricius, and the spleen. In adult birds neither the bursa nor the thymus make a direct contribution to production of effector molecules such as antibodies (74); these organs, however, appear to act as sites of cell maturation or provide influences essential for differentiation of precursor cells into immunologically active cells. The spleen of a normal chicken contains lymphoid follicles composed of large and medium lymphocytes, reticular cells, and a few dividing cells (72). The follicles are surrounded by cuffs of small lymphocytes. If the bursa is removed from a newly hatched chick and the bird is irradiated to ablate preexisting lymphoid cells, the recovered birds have hypogammaglobulinemia and cannot synthesize antibodies. Spleens from these birds do not have germinal centers, and plasma cells cannot be found. Cellular immune responses such as graft rejection and delayed allergy remain intact. This defect is analogous to the Bruton type of hypogammaglobulinemia, a sex-linked recessive human disorder of immunoglobulin synthesis. In contrast, neonatal extirpation of the thymus and irradiation of the chick produces a defect that is limited to cell-mediated immune responses. Antibody synthesis in these birds is normal, but they have defective rejection of allografts and delayed hypersensitivity reactions. Examination of spleens from K/rfcpatr/ck et a/. Chronic Mucocutaneous Candidiasis 963

10 Table 4. Serologic Responses In Patients with Mucocutaneous Candidiasis Patient Candida Antibodies* Immunoglobulins* Serum Serum Parotid Fluid Serum Parotid Fluid Agglutinins f Precipitins Agglutinins f IgG IgA IgM IgA < mg/ml ng/mg protein S.H. 4, ND J.C D.L. 1, M.L R.H. 2, G.R ND ND W.M R.M S.M. 1, H.S. 1, ND ND ND J.B. 4, K.B. 2, Normal range Negative * ND - test not done, t Reciprocal of titer. these birds discloses plasma cells in the germinal centers, but the cuffs of small lymphocytes are essentially absent. The immunologic defect in these birds is analogous to that found in children with congenital absence of the thymus, such as the Di- George syndrome (19). These observations prompted the two-component concept of differentiation of the immune response (75). According to this scheme (Figure 8), immunologically undifferentiated stem cells from the bone marrow receive differentiative directions from either the bursa or the thymus. Those under bursal influence populate the germinal follicles and respond to antigenic stimuli with synthesis of specific antibodies. Cells that differentiate under control of the thymus are found in the parafollicular areas and participate in cellular immune processes. As previously mentioned, the limited defects in patients with certain immune deficiency syndromes are compatible with this model. Presumably, patients with combined immunologic deficiency syndromes such as Swiss agammaglobulinemia, thymus dysplasia, or the Figure 8. The two-component hypothesis for differentiation of cellular and humoral immunity in the chicken. 964 June 1971 Annals of Internal Medicine Volume 74 Number 6 Wiskott-Aldrich syndrome have a defect at another site, perhaps in the bone marrow stem cell. Certain questions are not answered by the model. For example, with the possible exception of the rabbit (76), an anatomic counterpart of the bursa has not been identified in mammals. Furthermore, certain humoral responses in rodents appear to be dependent on normal thymus function (71). These points do not detract from the value of the model, however, as a framework from which critical questions regarding patients with abnormal immune responses may be studied. Host-Defense Mechanisms in Chronic Candidiasis A comprehensive survey of host-defense mechanism in patients with recurrent or chronic infections requires analysis of the complement system, of phagocytosis and killing of microorganisms by leukocytes, as well as investigation of the humoral and cellular immune responses. Seven patients with chronic mucocutaneous candidiasis had histories of repeated bacterial infections (Table 2). Each of these individuals and three additional patients with candidiasis had extensive studies of leukocyte functions. In no patient was there an abnormality in the capacity of polymorphonuclear leukocytes to ingest and kill Candida albicans (77) or bacteria (78). Phagocytosis induced normal stimulation of glucose oxidation via the pentose pathway (78), and histochemical stains for leukocyte peroxidase were always positive (77). Total hemolytic complement activity (79) was within the normal range (25 to 40 units) with the exception of two patients: K.B., with a titer of 44 units, and S.M., with a titer of 18 hemolytic units. In each of our patients, and in most cases re-

11 ported by others (63, 80), the humoral immune responses are intact (Table 4). Titers of Candida agglutinins and precipitins in the serum were usually greater than normal, whereas the parotid fluids contained normal antibody levels (29). Elevations of serum immunoglobulins were often found, and in two patients (R.H. and J.B.) unusually high concentrations of IgA were detected in the parotid fluids. Serum isoagglutinin titers were also normal. In all but two patients (M.L. and G.R.) intradermal skin testing with a Candida extract produced large wheal and flare reactions typical of those mediated bv reaginic antibodies. In contrast, abnormal cellular immune responses are common in patients with chronic mucocutaneous candidiasis, and reports from other laboratories have described negative skin tests to oidiomycin or other Candida extracts (63, 80). This observation is especially striking when one considers that approximately 80% of normal adult subjects have positive delayed skin tests to C. albicans (81, 82). In the investigations of cellular immunity we used a panel of antigens that included commercial and autologous Candida extracts (29), streptokinasestreptodornase (SK-SD), mumps antigen, purified protein derivative (PPD), and histoplasmin. The results of cutaneous testing permitted segregation of the patients into three groups (Table 5). Two individuals failed to respond to any antigen in the panel and appeared to have a generalized defect in cellular reactivity (group I). Patient R.H. is included in this group because his mumps skin test produced only equivocal induration. Neither R.H. nor J.C. could be sensitized with l-chlor-2,4 dinitrobenzene (CDNB). Group II was composed of six patients in whom the response to Candida was negative, but other antigens produced normal delayed reactions. One person, D.L., failed to respond to any natural antigens but is included in this group because he developed contact allergy after sensitization with CDNB. Finally, there were four patients in whom the reactions to Candida, as well as other antigens, were positive (group III). In summary, 8 of the 12 patients with candidiasis did not have delayed allergy to Candida extracts. In Vitro Studies of Cellular Immunity For many years evaluation of cellular hypersensitivity in humans has been limited to rather gross end points such as cutaneous reactivity or rejection of skin grafts. Recently, a number of in vitro systems have been developed that permit independent assays of the cellular and biochemical changes that follow exposure of sensitized lymphocytes to antigens. The most widely employed technique for studying delayed hypersensitivity in vitro is antigen-induced lymphocyte transformation (83). This term originally referred to morphologic changes and cell division in cultures of antigen-stimulated cells but has been extended to include a number of associated biochemical events such as increases in synthesis of DNA, RNA, and proteins. These antigen-induced in Table 5. Delayed Cutaneous Hypersensitivity Responses in Patients with Chronic Mucocutaneous Candidiasis Group Patient Antigen* I Candida Intermediate Histo Mumps SK-SD albicans PPD plasmin T" cm induration in *4 nv No response to any antigen R.H (?) 0 J.C II Response to antigen other than Candida D.L M.L R.M NDf W.M ND S.M K.B Ill Response to Candida albicans S.H G.R H.S J.B * Candida albicans extract was a sonicate of yeast phase organisms containing 1.0 fig protein in a skin-test dose. PPD = purified protein derivative; SK-SD = streptokinase-streptodoraase (Varidase, Lederle), containing 40 units SK and 10 units SD in a skin-test dose, t ND = test not done. Klrkpatrick et a/. Chronic Mucocutaneous Candidiasis 965

12 Table 6. In Vitro Lymphocyte Transformation in Chronic Mucocutaneous Candidiasis Group Patient Baseline* Candida albicans, 5 /xg/ml PHA-Mt0.05ml dpmf X 10~ 8 dpmf X 10" 3 Stimulation dpmf X 10~ 8 Stimulation Ratio Ratio I R.H J.C II D.L ND M.L R.M W.M S.M K.B III S.H G.R H.S ND Controls J.B Candida skin-test negative Candida skin-test positive * Baseline = dpm x 10-3 in tubes containing lymphocytes but no stimulating agent. t Dpm = disintegration per minute. X PHA-M = phytohemagglutinin-m (Difco). ND = test not done. vitro responses appear to be highly specific and in most situations correlate closely with delayed cutaneous hypersensitivity. In our studies incorporation of 3 H-thymidine into DNA has been used to quantitiate the phytohemagglutinin (PHA) and candida-induced responses of lymphocytes from healthy subjects with either positive or negative delayed allergy to Candida and to compare these values with patients with chronic candidiasis. The results of this investigation are summarized in Table 6. Some healthy individuals with negative Candida skin tests showed a small increment in DNA synthesis when Candida extracts were added to their lymphocytes in vitro. Healthy skin-test positive persons produced marked responses, with at least fourfold increases in incorporation of radioactive thymidine. Patients with chronic candidiasis with positive skin tests also produced brisk responses to stimulation with Candida albicans. A particularly provocative group was that composed of the chronic candidiasis patients with negative delayed allergy to C. albicans. Although most of these patients were poor responders, two individuals, R.H. and K.B., showed normal lymphocyte stimulation. Tentatively, this finding is interpreted as indicating that these patients comprise a unique subgroup that will be discussed in more detail later. Another technique for studying delayed hypersensitivity in vitro involves assaying the culture fluids from antigen-stimulated lymphocytes for certain biological activities such as chemotaxis, cytotoxicity, inhibition of migration of macrophages, and recruitment of nonsensitized cells into DNA synthesis (84). Production of these presumed mediator substances only occurs when sensitized cells are exposed to antigens and therefore is believed to depend on an interaction between the antigen and a specific cell receptor. The contribution of each substance to the final lesion of cellular hypersensitivity in vivo is not known; however, it has been shown that fluids from antigen-stimulated cultures, which presumably contain all of the "mediator" substances, produced typical delayed hypersensitivity responses after intradermal injection into guinea pigs (85). MEDIATOR PRODUCTION Our studies of mediator production have been limited to measurements of macrophage migration inhibition factor (MIF). This substance is produced by sensitized lymphocytes after exposure to antigens and prevents migration of guinea pig macrophages from capillary tubes. The areas of the fields of migration are measured with a planimeter. Figure 9 illustrates the results of a typical test. In fields A and B are shown the fans of migrating macrophages in fluids from lymphocyte cultures from a skin-test negative individual without (^4) or with (B) a C. albicans extract. There is no significant difference in the areas of the two fans. Fields C and D show the results of a similar test using lymphocytes from an individual with a positive Candida skin test. The area of migration in the fluids from antigen-stimulated cultures (D) was 31% less than that in fluids from antigen-free cultures (C). Reduction of the area of migration by 20% is significant (P < 0.02). Addition of antigen alone to culture fluids does not affect the migration of the macrophages. Production of MIF has been measured in seven 966 June 1971 Annals of Internal Medicine Volume 74 Number 6

13 A Model of Defects in Cellular Immunity Figure 9. The macrophage migration inhibition factor assay. Panels A and B illustrate results with culture fluids from lymphocytes from a Candida skin-test negative individual. There was no significant difference in the areas of migration in the absence (A) or presence (B) of the Candida antigen. A representative test with lymphocytes from a Candida skin-test positive individual is shown in panels C and D. In this case the area of migration in fluids from antigen-stimulated lymphocytes (D) was 31% less than the unstimulated control (C). patients with candidiasis (Table 7). Only two had positive skin tests; one produced MIF, and the second (H.S.) has repeatedly been negative. None of the five patients with negative skin tests have produced MIF when cultured with Candida antigen. One patient (K.B.) had a reactive skin test to SK-SD, and supernatants from lymphocytes stimulated with that antigen reduced migration by 24%. The results of the studies of cellular immunity in the patients with candidiasis may be summarized as follows: Four patients had positive skin tests to Candida, and each had a normal in vitro proliferative response to stimulation with Candida. Of the two patients in this group studied, one failed to produce MIF. In eight patients the Candida skin tests were negative, and two of these patients did not respond to any antigen in the panel. Of the five patients studied, none made macrophage migration inhibition factor. Two patients with negative skin tests and failure to produce MIF were unique in that they had normal in vitro proliferative responses. At the beginning of this presentation certain physiological requirements for immune competence were described. Although many issues remain to be settled, a general theory of the immune response may be summarized as follows: the precursors of immunologically reactive cells derive from the bone marrow (Figure 10). Cells that are to participate in cellular (delayed) hypersensitivity responses and certain humoral responses differentiate into antigenreactive cells under the influence of the thymus. Antigen-reactive cells have morphological characteristics of small lymphocytes and the physiological property of combining with antigen, presumably by way of a specific receptor on the cell membrane. The role of macrophages is not clear, but they may "process" the antigen into a critical unit or present antigens to antigen-reactive cells in a more efficient form (86). The chemical nature of the antigen receptor is unknown and is currently the subject of considerable debate. It has functional properties such as specificity, however, that are analogous to antibody molecules. Antigen-receptor interactions convey a "stimulus" to the cell, the nature of which is essentially unknown. When interactions lead to antibody production, the antigen-sensitive cell in turn interacts with other lymphoid cells, and it is these cells that actually synthesize immunoglobulins (87). The nature and number of cells involved in the sequence of reactions for delayed hypersensitivity are unknown, and this is also an area of intense study. In cellular immune reactions at least three responses may be observed after exposure of an antigen-reactive cell to antigen (Figure 10). Antigen-induced division of lymphocytes may be monitored in vitro by measuring incorporation of radioactive Table 7. Macrophage Migration Inhibition Factor (MIF) Production in Chronic Mucocutaneous Candidiasis Group Patient Areas of Migration Antigen- Candida Reduction Free Control albicans planimeter units % I R.H J.C II D.L S.M K.B III S.H H.S Controls Skin-test negative <10 Skin-test positive >20 Kirkpatrick et al. Chronic Mucocutaneous Candidiasis 967

14 Figure 10. Model of events leading to generation of cellular immunity in man. Postulated sites of defects that may be associated with candidiasis are identified by the numerals. precursors into cellular DNA. This response is apparently not of great magnitude at the site of the lesion because mitotic figures are rarely found. Replicative responses probably serve to expand the population of specific antigen-reactive cells to participate in anamnestic responses. Second, lymphocytes that have been "activated" by exposure to antigens or mitogens function as "aggressor cells" that injure or kill "target cells" by a process that requires direct contact (88). Rejection of grafts, graft-versus-host reactions, and some "autoimmune" disorders may be examples of the effects of aggressor cells. Third, mediators produced by antigen-stimulated lymphocytes have diverse properties, including chemotaxis, cytotoxicity, inhibition of macrophage migration, and recruitment of cells into DNA synthesis. Mediator synthesis occurs before DNA replication and independently of cell division. It is not known if mediators are produced by the same cells that eventually divide or by a separate, static pool of small lymphocytes. In any case, evidence has been provided that the mediators produce lesions histologically similar to delayed hypersensitivity reactions (85). From the scheme in Figure 10, one may identify specific abnormalities that occur in certain immune deficiency syndromes as well is in some patients with chronic candidiasis. A defect in generation or differentiation of lymphoid stem cells (site 1) would produce the combined immunologic deficiency syndromes. Children born with no thymus (site 2) have a defect in delayed hypersensitivity that apparently results from failure to differentiate antigen-reactive cells, and it has been mentioned earlier that these patients regularly have candidiasis. In terms of the postulated model, the six patients with candidiasis who failed to respond to Candida albicans with lymphocyte transformation, mediator production, or delayed cutaneous hypersensitivity are interpreted as having either defective function or absence of specific antigen-reactive cells (site 3). The basis of the defect is unknown, and both congenital and acquired mechanisms should be considered. The early age of onset of candidiasis may indicate a congenital defect. None of our patients, however, had studies of delayed allergy at the time of onset of the infection. Under certain conditions administration of an antigen may alter the normal response to a subsequent immunization. For example, guinea pigs immunized with soluble or alum-precipitated proteins produced serum antibodies but failed to develop delayed hypersensitivity when reimmunized with the same antigen in adjuvant. This phenomenon is known as immune deviation (89). A similar impairment in delayed hypersensitivity has been produced in animals given intravenous injections of antigen followed by injections of antibody to that antigen (90). It is unknown if the dose and route of exposure of the patients with candidiasis to Candida antigens is in any way analogous to these models, but the immunologic consequences appear quite similar. The reduction of lymphocyte transformation and MIF production in our patients is not owing to simple inactivation of the antigen in the cultures. Serum from patients with candidiasis does not reduce in vitro proliferation of lymphocytes from skin-test positive individuals, and lymphocytes from patients with candidiasis still do not transform when cultured in antibody-free serum. The MIF assay is performed in the absence of serum. Two patients with candidiasis had unique findings of normal in vitro transformation but defective expression of delayed hypersensitivity via skin tests and mediator production. According to the model, these patients possess antigen-reactive cells but are unable to differentiate cells that produce mediators (site 5). A patient with a similar abnormality has recently been described by Valdimarsson and colleagues (91). Our studies have not discovered patients with normal mediator production but impaired replication (site 4), and there are no studies of possible abnormalities in differentiation of function of aggressor cells. In summary, abnormal delayed hypersensitivity has been found in 8 of 12 patients with chronic 968 June 1971 Annals of Internal Medicine Volume 74 Number 6

15 mucocutaneous candidiasis. Tentatively, two types of cellular dysfunctions have been identified. Recognition and characterization of the specific defects in this heterogeneous population of patients may provide a rational basis for specific "defect-oriented" therapy. Prospects for Host-Directed Therapy Dr. Rich: The use of chemotherapeutic agents in the treatment of chronic mucocutaneous candidiasis has been previously outlined. Drugs are clearly useful in control of the disease and can, in fact, often produce complete clearing. Nonetheless, treatment directed only to the organism could predictably be inadequate, since a "fertile soil" for Candida persists in these deficient hosts even after elimination of the organisms. Consequently, although remissions with chemotherapy may be complete, they are almost always temporary. Specific defects in cellular immunity have been characterized in certain of these patients. If a pathogenetic role can be assumed for these defects in the establishment of candidiasis, a rational approach to therapy directed primarily to the host rather than the fungus can be developed. Tentative steps have been taken by investigators interested in reconstitution of patients with cellular immune deficiency syndromes. Spectacular therapeutic successes, as well as disastrous but instructive failures, have resulted from these efforts. The goals of reconstitutive therapy can be simply stated. Clinical cure with permanent correction of the immunologic defect is, of course, the primary goal. Failing this, one would hope for a substantial remission based on temporary improvement in immune function. Such remissions might achieve an effect of permanent cure, however, if the mode of therapy chosen permits repeated, possibly indefinite, application. Finally, these efforts should be designed, to an extent consistent with the patients' best interests, to extend understanding of human cellular immunity. The hazards of host-directed therapy can be substantial. Infusion or transplantation of cells and plasma is often used. In these instances the usual risks of transfusion must be assumed, primarily hepatitis and adverse antibody-mediated immunologic reactions. The latter deserves particular note, since these patients may be candidates for repeated transfusions. The risk of hepatitis, a potentially disastrous illness in certain immunologically deprived hosts (73), can be minimized by careful donor screening. The greater risk of immunologic reconstitution is the possibility of inducing graft-versus-host disease. Transfusion of patients who suffer profound immunologic incompetence with a population of immunocompetent cells may result in a reverse homograft rejection reaction (92). The grafted cells literally mount an immunologic attack against host tissues; the outcome is often death of the host. Fortunately, the likelihood of serious human graft-versus-host disease can.be reduced by close attention to the host and donor factors that promote its development. First, it is important to identify the population at risk. The hazard is probably minimal in adolescents and young adults with chronic mucocutaneous candidiasis. Although specific defects in cellular immunity can be identified in some of these patients, their cellular immune deficiencies are not complete, and certain critical functions are preserved. On the other hand, infants with profound morphologic abnormalities of the thymus gland, with or without associated stem cell deficiencies, are at great risk when given allogenic immunocompetent tissue and typically do very poorly. Certain studies are helpful in identifying that group of patients who lack the capacity to reject allogenic tissue (73). Except for those with the DiGeorge syndrome, these patients are usually lymphopenia Perhaps the most direct demonstration of an impaired capacity to deal with homografts is the failure of these patients to reject allogenic skin grafts. In vitro studies of lymphocyte function in these cases show that they do not proliferate in the presence of phytohemagglutinin. Options for Reconstitutive Therapy Several therapeutic alternatives are presently available for modification of human cellular immune function. Hematopoietic stem cells, with precursors for both humoral and cellular immunity, reside within bone marrow. Consequently, this tissue has been used for reconstitution of patients with combined stem cell defects (93-100). The risk of graft-versushost disease is substantially reduced if only tissue compatible at the major human histocompatibility locus, HL-A, is transplanted (101, 102). Simple Mendelian rules govern transmission of alleles at the HL-A subloci, and there is consequently a l-in-4 chance that any particular pair of siblings is HL-Aidentical (103). A dilemma obviously is faced when reconstitution with a stem cell population is advisable but no HL-A-identical sibling is available as a marrow donor. An alternative in this situation is transplantation of fetal hematopoietic tissue such as bone marrow (104) or liver ( ). The incidence of graft-versus-host disease is markedly reduced with these tissues, presumably by induction of graft Klrkpatrlok et at. Chronic Mucocutaneous Candidiasis 969

16 tolerance for host tissue antigens before differentiation of immunologic competence. Both fetal (94, 97, 104, 106, 108, ) and postnatal (115, 116) thymus tissue has also been transplanted. This may be adequate in cases with isolated thymus defects but normal marrow stem cell populations, for example, the DiGeorge (113, 114) and Nezelof-Allibone (21, 111) syndromes. Thymus transplants may provide the necessary microenvironment to allow differentiation of cellular immune function. Obviously, since stem cells are not provided by thymus transplantation, success would not be expected when treating primary stem cell disease with this tissue alone. The risk of graft-versus-host disease is clearly reduced when thymus rather than bone marrow is transplanted. Chimerism is not established with thymus grafting, however, and HL-A-identical thymus tissue has not been available. It is consequently possible that after restoration of host immunocompetence such grafts may eventually be rejected. Allogenic thymus is definitely less efficacious than syngenic thymus in reconstitution of thymectomized mice (117), and late rejection of allogenic thymus grafts correlating with deterioration of immune function has been observed in mice and postulated in humans (118). Peripheral blood lymphocytes offer an additional therapeutic modality. These cells are easily obtained in large numbers, and a full complement of differentiated immunocompetent cells, including antigenreactive cells and effector cells, can be provided. It is doubtful that permanent chimerism is often established by this means. In a compatible environment, however, a subgroup of human small peripheral lymphocytes may survive for years (119). It should be noted that peripheral blood lymphocytes are also capable of producing graft-versus-host disease, and a single transfusion has induced such a reaction, with fatal consequences, in susceptible recipients (106, 107, 109, ). The risk can be obviated by prior irradiation of the blood with at least 3,000 R when transfusion of susceptible patients for nonreconstitutive purposes is required (92). TRANSFER FACTOR A final reconstitutive reagent that has been used is a cell extract known as transfer factor. This material, originally described by Lawrence (123), is derived from lymphocytes of individuals with specific delayed hypersensitivity. It may transfer reactivity to the same antigen when administered to previously skin-test negative recipients. Transfer factor can be obtained from sensitized lymphocytes either by lysis or in vitro exposure of cells to antigen for 1 hour. Its chemical nature is not yet known, but it is resistant to digestion by ribonuclease, deoxyribonuclease, and trypsin. It is heat labile at 56 C and does not appear to be an immunoglobulin fragment. The molecule is relatively small; it will pass through a dialysis membrane and is thought to have a molecular weight of less than 10,000. The potential advantages of such a preparation over viable immunocompetent cells are apparent. There is no risk of graft-versus-host disease, and the dialyzed, deoxyribonuclease-treated material is free of cellular macromolecular components, is nonantigenic, and theoretically can be administered repeatedly. At present, essentially nothing is known about its mechanism of action. Most studies have been conducted in immunologically normal individuals, and it is probably premature to directly translate these observations to patients with ill-defined immunologic defects. It is conceivable, for example, that the material activates only one of the cell types in the scheme of cellular immunity depicted in Figure 10. If so, a patient with deficiency of that cell type might be expected to be refractory to transfer of delayed hypersensitivity by this means. In this context, transfer factor may have value as a reagent in definition of cellular immune defects. An obstacle to study of the transfer factor phenomenon is that it is apparently confined to primate species (123, 124). In contrast with other mediators of delayed hypersensitivity, which have been studied extensively in guinea pigs, efforts even to reproduce the observations in subprimates have been unsuccessful. Experience With Immunologic Reconstitution These are the therapeutic tools that have been used to date in attempts at reconstitution of human cellular immune deficiency. It is instructive to review reported experiences with these agents. Several investigators have undertaken immunologic treatment of patients with the lymphopenic agammaglobulinemias (Table 8). Three probably distinct diseases are included in this designation, namely Swiss-type agammaglobulinemia (22), thymus dysplasia (22), and thymic hypoplasia (100). They are considered together because all have a stem cell or combined immunologic defect. The therapeutic problems in each case are the same: a stem cell population is required for immunologic reconstitution, they are at great risk for graft-versus-host disease, and, untreated, the diseases have been universally fatal. The immunologic therapies attempted to date have been classified into four basic groups. At least 10 patients with these diseases have received allogenic peripheral blood lymphocytes (93, , 109, 110, , 125), not always as part of reconstitutive ef- 970 June 1971 Annals of Internal Medicine Volume 74 Number 6

17 Table 8. Summary of Experience with Immunologic Reconstitution of Patients with Lymphopenic Agammaglobulinemias Treatment Chimerism Laboratory Clinical Possible Death, Death, Mortality Reconstitution Reconstitution Cure GVH* Other Peripheral blood %~ lymphocytes Thymus or fetal 2/10 1/10 1/lOf 0/10 8/10 2/ hematopoietic tissues, or both 1/10 6/10 0/10 0/10 0/10 10/ Unmatched bone marrow 1/4 1/4 0/4 0/4 3/4 1/4 100 HL-A-matched bone marrow 2 or 3/3 3/3 3/3 f 3/3 0/31 0/3 0 * Graft-versus-host. t Candidiasis cleared. t Mild graft-versus-host disease with recovery. fort. One of these patients also received bone marrow (93), and three received fetal tissue (105, 106, 110). Chimerism was established in two cases, with XX karyotypes present in male recipients after prenatal, intrauterine engraftment with maternal blood (110, 125). One other patient developed evidence of immunologic reconstitution, with amelioration of lymphopenia and rejection of a previously established skin graft (106). This was accompanied by clearing of the associated candidiasis. Unfortunately, the outcome was unfavorable in every case. The HL- A-matched white cells were not used, and all 10 patients died, 8 of them with graft-versus-host disease. Thymus or fetal hematopoietic tissue without adult bone marrow, or both, or peripheral blood has been given to 10 patients with lymphopenic agammaglobulinemia (104, 108, 109, 112, 115, 116). Chimerism was established in one recipient of fetal liver (108). Six patients demonstrated some evidence of reconstitution, with synthesis of a monoclonal immunoglobulin by one (112), development of delayed hypersensitivity by one (116), and increased lymphocyte counts in four patients (104, 108). None of these patients was clinically improved, however, and, although none developed graft-versus-host disease, the mortality rate was again 100%. Four patients with lymphopenic agammaglobulinemia have been treated with HL-A nonidentical bone marrow (93-95, 98). All of these patients died, at least three with graft-versus-host disease. Chimerism was documented in one case (95), and one developed delayed hypersensitivity and remission of lymphopenia before death (94). Finally, three patients with stem cell defects have been recipients of HL-A-matched bone marrow (96, 97, 99, 100). One of these also received fetal thymus tissue (97), and one also received peripheral blood cells from the bone marrow donor (96). The experience reported with these patients has been genuinely exciting. Each of the recipients has shown broad evidence of immunologic reconstitution, with improvement in several of the usual measures of immunocompetence. Candidiasis cleared in each case. Chimerism was clearly established in two patients (96, 97) and probably in the third, although antigenic or chromosomal markers were not available in the latter instance. Two of the patients have required second transplants (99, 100). Although all three had evidence of graft-versus-host disease, in each case this was relatively mild, and the patients seem to have recovered. Long-range follow-up of these three patients will, of course, be of extreme interest. They may represent permanent cures of a preyiously fatal illness. Two patients with the DiGeorge syndrome have been treated by transplantation of fetal thymus (113, 114). In contrast to patients with the lymphopenic agammaglobulinemias who received only this tissue, both of these patients appear to have had favorable results. Candidiasis has cleared, and there has been good evidence of improved cellular immune function, including development of delayed hypersensitivity, skin graft rejection, and a blastogenic response of cultured lymphocytes to phytohemagglutinin. Further study of these patients, with particular attention to late graft rejection and immunologic deterioration, will be of interest (73). One patient with the Allibone-Nezelof syndrome was treated several years ago with blood, fetal bone marrow, spleen, and thymus tissue (111). Little if any clinical improvement was achieved, however, and the patient died of progressive vaccinia. Another patient with this disorder received transfusions of viable leukocytes and died 16 days later of graftversus-host disease (121). OTHER SYNDROMES We now digress briefly to mention two patients Kirkpatrick et al. Chronic Mucocutaneous Candidiasis 971

18 Table 9. Results of Immunotherapy of Chronic Mucocutaneous Candidiasis In Patients with Negative Candida Skin Tests and Unknown in Vitro Proliferative Responses Reference Number 34 Paternal bone marrow and amphotericin B X 10 9 peripheral blood lymphocytes Skin Test Conversion Migration Inhibition Factor Production Clinical Response yr posttherapy >2J? Candidiasis cleared No? None without candidiasis but of considerable interest to those concerned with reconstitutive therapy. These are patients with the Wiskott-Aldrich syndrome, a complex and poorly understood disease with sexlinked recessive inheritance. It is characterized by eczema, recurrent infections, severe thrombocytopenia with bleeding, and defects in both humoral and cellular immune function (126, 127). One of these patients was treated with HL-A-identical marrow on two occasions, the second time after pretreatment with cyclophosphamide (128). Chimerism was established, and striking improvements in both laboratory and clinical variables were observed (128) and maintained for at least 22 months (129). A second patient, recently reported (129), was treated with transfer factor. Transfer of delayed hypersensitivity was achieved, and the patient has apparently improved in both immunologic and nonimmunologic functions. CANDIDIASIS PATIENTS WITH NONLETHAL CELLU LAR IMMUNE DEFECTS The final group of patients to be considered is that which includes the series we are reporting, that is, patients with nonlethal cellular immune defects and chronic mucocutaneous candidiasis. All of these patients receiving therapy directed to reconstitution, both by ourselves and others, have had negative skin tests to Candida antigens before treatment. As previously noted, we regard the presence or absence of an in vitro proliferative response by lymphocytes cultured with Candida antigen to be useful in further characterizing the defect in these patients. In earlier reports the response to Candida antigen in vitro was not known (Table 9). A dramatic response was achieved, however, in one patient treated with paternal bone marrow infusion and intravenous amphotericin B (34). Although chimerism probably was not achieved, the patient did develop a positive intradermal skin test that was maintained for at least 2V2 years. More important, nearly complete clinical remission for the same period was also achieved. No effect was noted in a second patient treated with a small number of peripheral blood lymphocytes (130). We have treated two skin-test negative patients who had no proliferative response to Candida antigen in vitro (Table 10). Both of these patients, however, appeared to have selective defects for Candida antigen; they had normal responses in vitro and positive skin tests with other antigens. This suggests a specific absence of functional antigen-reactive cells for Candida antigens. The first of these patients was injected intramuscularly with a centrifuged and milliporefiltered lysate of 600 X 10 6 peripheral blood lymphocytes from a skin-test positive donor (29). Skin test conversion never developed, and no clinical response was apparent, but 30 days after treatment the patient did have a normal proliferative response in vitro. Thymidine incorporation by lymphocytes exposed to Candida antigens was 5.6 times that of unstimulated cells. A second of our patients is a 27-year-old woman with a negative Candida skin test, an absent in vitro proliferative response, and a failure of lymphocytes cultured with Candida antigen to synthesize macro- Table 10. Results of Immunotherapy of Chronic Mucocutaneous Candidiasis In Patients with Negative Candida Skin Tests and Negative In Vitro Proliferative Responses Reference Number Treatment Skin Test Conversion Conversion of Proliferative Response Migration Inhibition Factor Production Clinical Response 29 Cell lysate 59.4 X 10 9 peripheral blood lymphocytes No No days posttherapy 30 No? No None None 972 June 1971 Annals of Internal Medicine Volume 74 Number 6

19 Rgure 11. Remission of chronic cutaneous candidiasis after transfusions of allogeneic lymphocytes. A. Note numerous papular lesions over the nape of the neck. B. Essentially complete clearing of the neck lesions occurred by the third week after the transfusions. phage migration inhibition factor. This patient was infused over 4 days with HL-A-nonidentical fraternal leukocytes obtained via an NCI-IBM blood cell separator. A total of 59.4 X 10 9 lymphocytes were transfused. Karyotype of circulating lymphocytes was studied twice 24 hr after the last transfusion. On both occasions the patient was 60% XX and 40% XY. Nevertheless, neither at this time, when large numbers of viable fraternal cells were circulating, nor at any later date did the patient develop a positive skin test, exhibit a proliferative response in vitro, or synthesize MIF in response to Candida antigen stimulation. Clinical improvement was not observed. We have obtained substantially different results in a patient with anergy and a failure to synthesize MIF but with an essentially normal proliferative response of lymphocytes cultured with Candida antigen (Table 11). This patient received 63.6 X 10 9 paternal peripheral blood lymphocytes, also obtained with an NCI-IBM cell separator (27). Before transfusion the patient had only 0.6 cm of erythema and soft induration 24 hr after a mumps skin test. There was no delayed hypersensitivity to tuberculin, histoplasmin, SK- SD, or Candida antigen, and the patient could not be actively sensitized with topical CDNB. Twenty-four hours after transfusion, however, passive transfer of delayed hypersensitivity from paternal responses including mumps, Candida, SK-SD, and CDNB were observed. This cutaneous reactivity was maintained for at least 190 days. An attempt to detect MIF Table 11. Results of Immunotherapy of Chronic Mucocutaneous Candidiasis in Patients with Negative Skin Tests and Positive in Vitro Proliferative Responses Reference Treatment Skin Test Migration Clinical Number Conversion Inhibition Response Factor Production * Same patient. t Purified protein derivative X 10 9 peripheral blood lymphocytes 2 X 10 8 peripheral blood lymphocytes from skin-test negative donor 2 X 10 8 peripheral blood lymphocytes from skin-test positive donor* Transfer factor* Transfer factor < days postthei rapy > Candida and PPDf skin tests 6 Candida skin test only No 190?? 6 No Progressive clearing 220 days, relapse at 315 days None None None None Kirkpatrick et al. Chronic Mucocutaneous Candidiasis 973

20 synthesis shortly after transfusion was a technical failure. At 220 days, however, inhibitory activity of supernatants from the patient's antigen-stimulated lymphocyte cultures was essentially identical with a skin-test positive control, with 21% inhibition of migration. During this period gradual but definite improvement in the patient's candidiasis was observed. Nearly complete clearing of lesions, except those of hands and feet, was noted (Figure 11). Response of the more severely involved distal extremities was much less dramatic but probably also significant (Figure 12). The patient was again seen at 315 days in clinical relapse. Previously cleared areas were again infected, and, of great interest, the patient no longer maintained his passively transferred delayed hypersensitivity and was again unable to synthesize MIF. Other investigators have also treated several patients with probable defects in mediator synthesis (131, 132), that is, an in vitro proliferative response with Candida antigen but a negative skin test and failure to synthesize MIF. Our experience is summarized on the top line of Table 11. In none of the cases, except that which we have reported, was a significant clinical response observed. Figure 12. Remission of chronic candidiasis after transfusions of allogeneic lymphocytes. This picture was taken 6 months after the transfusions and should be compared to Figure 2B. Note the decrease in size of the cutaneous horn and partial regression of lesions over the dorsa of the feet. Interestingly, however, the results do not support the contention that transfusion of HL-A-nonidentical lymphocytes and transfer factor administration constitute essentially equivalent therapy (133). Note first that active sensitization of transfused lymphocytes obtained from a skin-test negative donor has been possible at an appropriate interval after lymphocyte injection (131). Another patient has received both a lymphocyte injection and transfer factor therapy (131, 132). In both cases the donor had delayed hypersensitivity to both Candida and tuberculin antigens. In the first instance the patients converted both Candida with tuberculin skin tests at 24 hr and maintained delayed hypersensitivity for 14 days (131). Synthesis of MIF was not studied. After receiving transfer factor, however, only the Candida skid test became positive, and this only transiently at 6 days (132). Production of MIF was also observed at this time. One other patient with this syndrome has been the recipient of transfer factor from a skin-test positive donor (132). In this case no effect on delayed cutaneous reactivity or mediator synthesis was noted. PROSPECTS This is the experience to date with immunologic reconstitution. What conclusions are presently reasonable on the basis of these reports? What are the prospects for host-directed therapy? Only partial and tentative answers can yet be provided. In the instance of patients with the lymphopenic agammaglobulinemias, HL-A-identical bone marrow transplantation can apparently result in reconstitution to essentially normal immune function. Clinical improvement, including clearing of candidiasis, has been concomitant in each case with laboratory reconstitution. In instances in which an HL-A-matched donor is not available, treatment with fetal hematopoietic tissue seems reasonable. Although no therapeutic successes have yet been achieved, this treatment does have a rational basis, and death from graft-versus-host disease has not been reported. It is apparent that these patients should no longer be recipients of unmatched bone marrow or viable peripheral blood lymphocytes. The disastrous effect of lethal graft-versus-host disease has become the expected result. Thymus transplantation of patients with lymphopenic agammaglobulinemias cannot be expected to reconstitute these patients, since these appear to be stem cell diseases rather than simply disorders of the thymic-dependent environment. On the other hand, early experience suggests that thymus transplantation is useful in treatment of patients with the DiGeorge syndrome. Whether these 974 June 1971 Annals of Internal Medicine Volume 74 Number 6

21 patients should also receive fetal hematopoietic cells is not clear. Because this is a primary defect of the thymus gland, exposure to the hazards of totally immunocompetent cells, such as bone marrow or viable peripheral white cells, does not seem advisable, at least not before an attempt at reconstitution with thymus tissue. Whether human allogenic thymus transplantation will achieve permanent or only temporary restoration of these patients is as yet unknown. Conclusions cannot be drawn yet regarding the Wiskott-Aldrich syndrome. Two reports purport laboratory reconstitution and clinical improvement by considerably different means (128, 129). Substantially greater experience will be required before the merits of HL-A-identical bone marrow transplantation can be compared to the effect of transfer factor administration in this disease. Finally, it is apparent that the role of immunologic reconstitution in the treatment of patients with chronic mucocutaneous candidiasis but without lethal abnormalities of the thymus gland is not clear. In fact, whether this approach will ultimately have any therapeutic importance is still uncertain. Nonetheless, it has been demonstrated that correction of the cellular immune defect can be associated with regression of the disease (27, 34). The apparent cure of a patient by Buckley and colleagues (34) with paternal bone marrow plus intravenous amphotericin B suggests that a combination of chemotherapy and immunotherapy may be the advisable approach for future efforts. During this period of evaluation it seems obvious that host-directed therapy should be confined to patients with specifically characterized cellular immune defects. Moreover, the therapeutic approaches used should be relatively benign. For example, one could not advocate cyclophosphamide pretreatment before bone marrow administration in these patients with usually nonlethal disease. Lastly, one can hope that future therapeutic efforts will be rationally directed to correction of specific defects and will be done in such a manner that results of treatment can extend understanding of the pathogenesis of the disease and of basic concepts of human cellular immunity. Concluding Remarks Dr. Kirkpatrick: In closing, several points should be reemphasized. Chronic candidiasis may occur in a number of clinical settings and probably should be considered a complication of an underlying disorder rather than a primary disease. Of particular interest is the fact that chronic candidiasis usually occurs in patients with diseases or congenital defects that depress cellular immunity, or in recipients of immunosuppressive drugs. Among the chemotherapeutic agents, intravenous amphotericin B is the only agent that regularly produces remissions or regressions. Even with this drug, sustained clinical improvement is rare. Presumably, candidiasis recurs after cessation of therapy because of a persistent defect in host defenses that renders the patient susceptible to reinfection with this opportunistic agent. Nonetheless, there is an obvious need for additional drugs with little toxicity that may be administered orally for prolonged periods. In this presentation we have discussed an adjunctive mode of therapy, that is, correction of the underlying cellular immune defect of the host. Obviously, identification and precise characterization of the immunologic abnormality in each patient is essential before institution of investigative therapy. Past experiences have demonstrated the potential hazards of immunological restitution with allogenic tissues and have provided certain guidelines for future studies. ACKNOWLEDGMENTS: The studies of leukocyte phagocytic activity and metabolism were performed by Dr. Richard Root, Leukocyte Physiology, and Dr. Ira Mickenberg, formerly Clinical Associate; complement titers were done by Dr. Michael Frank, Pediatric Immunology; and Mr. T. K. Smith furnished excellent technical assistance; at the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Autoantibodies were determined by Dr. H. H. Fudenberg, University of California, San Francisco, Calif., or Dr. R. Blizzard, Johns Hopkins University, Baltimore, Md. Karotypes were done by Dr. J. Whang-Peng, Medicine Branch, National Cancer Institute, Bethesda, Md. Leukapheresis with the NCI-IBM cell separator was done under the direction of Dr. Robert Graw, Human Tumor Cell Biology Branch, National Cancer Institute. Leukocyte typing was done by Dr. Paul Terasaki, University of California, Los Angeles. Received 10 March 1971; accepted 22 March Requests for reprints should be addressed to Charles H. Kirkpatrick, M.D., Head, Clinical Allergy and Hypersensitivity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bldg. 10, Rm. ll-b-13, National Institutes of Health, Bethesda, Md References 1. AUSTWICK PKC, PEPIN GA, THOMPSON JC, et al: Candida albicans and other yeasts associated with animal disease, in Candida Infections, edited by WINNER HI, HURLEY R. Edinburgh, E. & S. Livingstone, Ltd., 1966, p COHEN R, ROTH FJ, DELGADO E, et al: Fungal flora of the normal human small and large intestine. New Eng J Med 280: , MARWIN RM: Relative incidence of Candida albicans on the skins of persons with and without skin disease. / Invest Derm 12: , NILSBY I, NORDEN A: Studies on the occurrence of Candida albicans. Acta Med Scand 133: , AJELLO L: Soil as natural reservoir for human pathogenic fungi. 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