Extracorporeal Photopheresis in the Treatment of AIDS-Related Complex: A Pilot Study Emil Bisaccia, MD; Carole Berger, PhD; and Albert S. Klainer, MD
Objective: To determine side effects of extracorporeal photopheresis in the treatment of patients with the acquired immunodeficiency syndrome (AIDS)-related complex, and to gain early evidence of efficacy of the treatment. Design: Uncontrolled trial. Setting: Tertiary referral center. Patients: Five patients with AIDS-related complex: three homosexual men and one man and one woman with histories of intravenous drug abuse. One patient, a homosexual man, withdrew from therapy after 5 months but returned for monthly clinical and laboratory evaluations. Intervention: Monthly treatments with extracorporeal photopheresis. Measurements and Main Results: Symptoms resolved in four patients. Lymphadenopathy disappeared in all five. Four patients had delayed-hypersensitivity reactions to skin testing (as defined by the Walter Reed staging classification). All showed increases in p24 and gpl20 antibody levels. The CD4-cell percentage increased in four patients and declined in one after 6 months of therapy, but the absolute CD4 count decreased in two patients. At 15 months, the CD4 percentage remained at or increased over the baseline value in three patients still in the study but decreased in one. Levels of Beta2-microglobulin decreased or remained stable in four patients. All patients were culture positive for the human immunodeficiency virus (HIV) before treatment. One patient had a negative viral culture after 5 months of treatment with confirmation. Two other patients became HIV culture negative, one at 14 and one at 15 months: The former patient became positive at 15 months and the latter patient remained negative at 16 months. Conclusions: The preliminary results suggest that extracorporeal photopheresis deserves further evaluation as therapy for AIDSrelated complex.
Annals of Internal Medicine. 1990;113:270-275. From Morristown Memorial Hospital, Morristown, New Jersey, and Columbia University College of Physicians and Surgeons, New York, New York. For current author addresses, see end of text. 270
© 1990 American College of Physicians
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I n vitro, virus-inactivating agents may function by modifying viral coats or damaging viral nucleic acid, or by doing both simultaneously. Photochemical inactivation of DNA viruses using psoralen derivatives was first demonstrated in vitro in 1961; the technique was later extended to RNA viruses. Irradiation of viruses with long-wavelength ultraviolet A in the presence of psoralen results in cross-linking of base-pair regions of nucleic acids (1, 2), with formation of covalent monoadducts. Both DNA and RNA viruses, including the human immunodeficiency virus (HIV), have been inactivated in vitro by psoralen photoreaction (3). Recently, in a study by Edelson and associates (4), patients with cutaneous T-cell lymphoma, a CD4-positive lymphocytic malignancy, showed a marked clinical response to photopheresis (4). In this therapy, 8-methoxypsoralen is administered orally before ultraviolet A irradiation of patients' leukocytes in an extracorporeal photopheresis device; after irradiation, cells are reinfused. The primary target for HIV infection is the CD4positive helper-inducer T-cell lymphocyte; the cytopathic consequences of the infection are most prominent in this lymphocytic cell population. Additional susceptible cell types include those of the macrophagemonocyte lineage. Human immunodeficiency virus infection of these cells in the peripheral blood, lymphatics, and other tissues has been well documented. The mechanism underlying the CD4 treated-cell depletion that occurs in vivo in HIV infection may involve cell fusion, indirect and undefined cytopathic processes, and possibly an autoimmune mechanism (5). Such depletion causes immunologic debilitation, which is ultimately fatal within 12 to 48 months as these patients progress from AIDS-related complex to AIDS. The terminal event is most commonly an infection (6), as is the case in patients with cutaneous T-cell lymphoma (4). The accessibility of the blood leukocytes and, therefore, the CD4-positive cells to an extracorporeal system involving psoralen and ultraviolet A light led to the consideration of a phase 1 trial in patients with HIV infection. Because no adequate animal model is available for studying HIV infection in vivo or for evaluating the response to new potential therapies, a trial was undertaken in humans. A pilot study was begun with five volunteers who had photopheresis on 2 consecutive days at monthly intervals. The results of the initial 6 months of phase 1 treatment are reported for five patients with AIDS-related complex; the study, having extended to 14 to 15 months, is now a phase 2 trial. A phase 1 study is designed to determine side effects and, if possible, to gain early evidence of effectiveness. A phase 2 trial is done to obtain a preliminary eval-
Table 1. Clinical Characteristics Characteristic Sex, age, y Predisposing factor Walter Reed staging classification Known duration of HIV antibody positivity, y Weight, kg Before treatment After treatment* Presentt Absolute lymphocyte count, /mm3 Before treatment After treatment Present CD4-positive cells, /mm3 (%) Before treatment After treatment Present CD4:CD8 ratio Before treatment After 6 months Present p24 antibody level (absorbance)§ Before treatment After 6 months Present gpl20 antibody level (absorbance)|| Before treatment After 6 months Present Virus culturell Before treatment After 6 months Present Beta2-microglobulin, mg/L Before treatment After treatment Present p24 antigen, pglmL Before treatment After treatment Present
and Laboratory
Results
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
M, 38
M, 30
M, 27
Homosexuality 4
Homosexuality 5
Homosexuality 5
M, 42 Intravenous drug abuse 4
F, 27 Intravenous drug abuse 5
3
2
4
2
1
71.3 70.5 71.0
64.5 66.0 68.7
63.6 64.7 64.5
2600 1768 1620
1824 1533 1554
1617 893 546
109 108 110 1024 2067 2176
61.0 57.7 56.0 1936 522 594
104 (4)t 601 (34) 181 (25)
529 (29) 475 (31) 451 (29)
550 (34) 330 (40) 282 (52)
113 (11) 682 (33) 587 (27)
232 (12) 68 (13) 42(7)
0.2 0.6 0.5
0.8 0.7 0.7
1.0 1.1 1.1
0.8 0.8 0.8
0.1 0.8 0.8
2.52 2.76 2.78
0.12 0.74 0.79
0.72 1.69 2.02
0.62 1.76 2.50
1.41 2.89 2.68
2.22 1.68 2.89
0.03 0.14 2.59
1.04 2.07 2.46
0.21 1.25 2.54
0.46 1.95 2.63
+ + +
+
+
+ +
-
+ + -
+ + +
NA 2.8 2.6
3.6 3.6 3.6
NA 4.3 3.1
3.9 4.3 NA
NA 495 258
NA
NA 135 54
NA
NA** 7.6 6.2 NA
-
-
-
* After 6 months of therapy except in Patient 3 who received only 5 months of therapy (see text). t After 14 to 15 months of therapy (see text). X Percentage of peripheral T cells that are CD4 positive. § Standard deviation for p24 antibody (± 13%). |j Standard deviation for gpl20 antibody (± 20%). 11 Culture was done using peripheral blood mononuclear cells. ** NA = not available.
uation of the efficacy for a particular indication. The preliminary results suggest that serious short-term toxicity is not an inherent feature of the photopheresis regimen. Four of the five patients had an encouraging response. Patients and Methods Five patients with AIDS-related complex were selected from the six patients who volunteered for the study. The patient who was not selected was disqualified because he was enrolled in another drug trial. All five patients had enlarged lymph nodes (1 cm or more) at two or more sites; and unexplained systemic symptoms, including intermittent or continuous fever for more than 1 month, repeated night sweats, a debilitating, persistent weight loss of at least 5%, and a decreased percentage of CD4-positive cells. The study was done according to the provisions for the treatment of human subjects of the Institu-
tional Review Board at Morristown Memorial Hospital, Morristown, New Jersey. The study group comprised three homosexual men who ranged in age from 27 to 39 years, one 42-year-old man with a history of intravenous drug abuse, and one 27-year-old woman with a history of intravenous drug abuse. One to four years had elapsed between the patients' first known HIV antibody positivity and their enrollment in the study (Table 1). Only patients who had not had any previous therapy for HIV infection were enrolled. The patients were interviewed by the principal investigators and answered a quality-of-life questionnaire (available from the authors on request). On the basis of the Walter Reed (WR) staging classification (7), patients ranged from WR4 to WR5 (Table 1). Cervical, supraclavicular, axillary, epitrochlear, and inguinal lymph nodes were palpated before enrollment and before each treatment by an experienced physician. Lymph node enlargement was defined as a palpable node of 1 cm or more at two
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Table 2. Lymph Node Examination Patient
Cervical Right Left
before
Treatment
Supraclavicular Right Left
Axillary Right Left
cm
1 2
2 1
3 1
... ...
... ...
1 1
3 4 5
... 1 1
... 1 1
... ... ...
... ... ...
... ... ...
or more sites. The size of the lymph node was approximated by the use of standard skin calipers (Table 2). Nonblinded skin testing with four standard recall antigens was done in the standard manner to induce a delayed-hypersensitivity reaction. A cutaneous response was considered positive if skin induration of at least 5 mm resulted from application of two or more of the following four test antigens: Trichophyton, mumps, Candida, and intermediate-strength tuberculin purified protein derivative (Table 3). Skin biopsy with histologic confirmation was obtained when indicated and was evaluated by a blinded observer. Pretreatment studies included an enzyme-linked immunosorbent assay (ELISA) for HIV and a confirmatory Western blot, antibody tests (HIVAGEN-SKF, SmithKline Beecham, West Norriton, Pennsylvania), HIV isolation by p24 antigen capture (Metpath, Teterboro, New Jersey), peripheral blood phenotyping, hepatitis profile, VDRL test, antinuclear antibody test, chest roentgenogram, and electrocardiogram. Pretreatment and monthly post-treatment studies included a complete blood count, platelet count, erythrocyte sedimentation rate, reticulocyte count, a 20-parameter automated analysis, and urinalysis. In month 3 of the trial, determinations of cytomegalovirus and Epstein-Barr virus titers, immunoglobulin electropheresis, and assessments of beta2-microglobulin and p24 antigen levels were added. Isolation of HIV was repeated every 3 months. All patients were weighed before enrollment and before each monthly treatment. Skin testing to the recall antigens of tuberculin, mumps, Candida, and Trichophyton was done before treatment and at the completion of approximately 6 months of therapy. Lymphocytes isolated from peripheral blood using FicollHypaque separation were stained with monoclonal antibodies, and the percentages of CD3-positive, CD4-positive, and CD8positive T cells were determined by flow cytometry (8). The absolute numbers of CD4-positive and CD8-positive T cells were calculated from the total and differential leukocyte counts. We measured beta2-microglobulin by radioimmunoassay (Pharmacia, Uppsala, Sweden) using standards provided by the manufacturer (9). Isolation of virus was done using a modification of the technique developed by the Centers for Disease Control (10). Lymphocytes obtained from heparin-treated whole blood were isolated by Ficoll-Hypaque separation, cultured, and assayed for p24 antigen at 7, 14, 21, and 28 days. Results of virus isolation were confirmed at 3-month intervals. We measured HIV p24 antigen using a solid-phase, sandwich-type enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, Illinois) (11). This assay uses a polyclonal HIV antibody and a purified viral lysate as a standard; therefore, p24 antigen and other viral proteins are measured. An assay was considered positive if the optical density of the culture supernatant was greater than that of the standard. The specificity of each positive specimen was confirmed by a neutralizing assay using 50 /ig of a polyclonal human anti-HIV IgG solution. A reduction of more than 50% of the recorded absorbance was considered to be confirmatory (11). We tested for HIV antibodies using the Western blot and HIVAGEN assays. The HIVAGEN assay is based on six purified recombinant antigens and is used to test for HIV antibodies in the screened sera. It has been suggested that this assay be used for longitudinal evaluation of seropositive patients for disease progression or antiviral therapy (11). 272
Epitrochlear Right Left
1 2
... ...
1.75 ... ...
1 ... ...
1
2 ... ...
3 3
3
2 3 3
Western blot assays were done according to the instructions of the manufacturer (Du Pont, Wilmington, Delaware). In our study, photopheresis was done using the UVAR instrument (Therakos, West Chester, Pennsylvania), which integrates an initial discontinuous leukapheresis step with exposure to ultraviolet light in a single apparatus. The procedure involved pooling 240 mL of leukocyte-enriched blood with 300 mL of the patient's plasma (removed 2 hours after ingestion of 0.4 to 0.6 mg/kg body weight of 8-methoxypsoralen) and 200 mL of sterile normal saline. The leukocyte-enriched fraction was obtained by centrifugation, using a 125-mL pediatric bowl and a six-cycle leukapheresis technique (UVAR instrument, Therakos, West Chester, Pennsylvania). The total volume was then passed as a 1-mm film through a sterile irradiation chamber to expose it to ultraviolet A energy delivered by a fluorescent source. After exposure of blood to ultraviolet A, the entire amount was returned to the patient (4). Each patient received a monthly treatment. Results Four patients received a total of 12 treatments each as part of the 6-month phase 1 study that has now continued through 14 to 15 months, evolving into a phase 2 trial. The fifth patient (Patient 3) voluntarily withdrew from the phase 1 study after 5 months of treatment (10 treatments) but continues to return for monthly clinical follow-up and laboratory evaluation. All patients had a positive Western blot, a positive test for antibody to two HIV-1 gene products, and a positive HIV culture. On the basis of the physical examination, clinical indices, monthly antibody tests, p24 antigen studies, and the percentage or absolute number of CD4-positive cells, four of five patients were judged to have had either a stabilization of their disease or an encouraging response to treatment (Table 1). The absence of significant toxicity during the treatment course was suggested by the stabilization of the percentage of CD4-positive cells, the CD4:CD8 ratio, or the absolute number of CD4-positive cells, and an increase in antibody titers (Table 1). On the basis of responses to the quality-of-life questionnaire and a review of symptoms in conjunction with evaluation of clinical status, four patients showed some improvement. Initially, the increase in energy lasted for approximately 1 week after treatment. By the third treatment, the increase in energy persisted, and the general quality of life improved. Most notably, the first patient treated, a homosexual man who before treatment was able to walk up a flight of stairs only with difficulty, is now capable of jogging 3.5 miles a day and lifting weights. In addition, two other participants have shown similar subjective improvement: Both are now attending college and working full-time. The one patient who did not show a persistent rise in energy level was
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3 2
... ...
study have shown either a stabilization of or an increase from pretreatment levels in the percentage and absolute numbers of CD4-positive cells and in the CD4.CD8 ratio. In these same three patients, the absolute lymphocyte count increased in one, was relatively stable in one, and decreased in one. The beta 2 -microglobulin levels decreased in two patients and remained relatively stable in the other (Table 1). The p24 and gpl20 antibody levels increased in all patients (Table 1).
the woman with a history of intravenous drug abuse who had three small children. Appetite increased and weight remained stable in all patients except this woman, whose weight decreased 3.3 kg during the 6month treatment period (Table 1). Fever and night sweats disappeared after 3 months of therapy. Lymphadenopathy (Table 2) disappeared in all patients by the third month of therapy and remained absent. Three patients were anergic in skin tests before treatment; two of these patients reacted positively to two skin-test antigens after therapy. The two remaining patients reacted to a single antigen before therapy and both responded to a second antigen after therapy, thereby meeting our case definition for positive skin-test reactivity (Table 3). In two patients, the positive skintest sites were biopsied, and delayed-hypersensitivity reactions were confirmed by a blinded observer. During the 15-month period of treatment, common communityacquired upper respiratory tract infections were managed in all patients without difficulty. Except for normal variations, the hemoglobin, hematocrit, reticulocyte count, and platelet count remained stable. There were minor variations in the leukocyte count. In all patients, erythrocyte sedimentation rate, results of urinalysis, and results of a 20-parameter automated analysis remained unchanged, except for minor elevations in aspartate aminotransferase and alanine aminotransferase. Epstein-Barr virus titers remained constant in all but one patient: The titer of his IgG capsid antigen decreased from 1:1280 to 1:160, which paralleled an increase in HIV antibody titers. Cytomegalovirus titers remained unchanged in all. At 6 months, those patients who were p24 antigen negative before treatment remained negative, and those who were positive remained positive with no significant changes in titer. By 15 months, the p24 antigen levels had decreased in the two antigen-positive patients (Table 1). Cultures of blood for HIV became negative in Patient 3 after the fifth treatment; this was confirmed twice during the ensuing 9 months. Two additional patients, Patients 1 and 4, became negative for HIV at 14 and 15 months, respectively. Patient 4 was still negative at 16 months; Patient 1, however, became positive at 15 months. During approximately 15 months of treatment, the three male patients who remained in the Table 3. Skin Testing for Delayed Test Trichophyton Before treatment After treatment* Tuberculin purified protein derivative (intermediate strength) Before treatment After treatment Mumps Before treatment After treatment Candida Before treatment After treatment
Discussion The paramount concern with the application of photopheresis in patients with HIV infection is that the reinfusion of T cells treated or activated by photopheresis would lead to further depletion of the CD4-positive population. Data from in-vitro studies have shown that a substantial loss of viability occurs over time in normal lymphocytes on exposure to 8-methoxypsoralen and ultraviolet A light (12). Approximately one third of the circulating lymphocytes are treated in the described methods, and the possible reduction of lymphocyte viability in patients with HIV infection might have resulted in severe harm to the patients. Because psoralen enters all nucleated cells, DNA adduct formation occurs in every cell exposed to the drug and ultraviolet A light. This cross-linking of DNA is not repairable by the cells' normal mechanisms and has potential implications for other cells of the immune system (for example, monocytes, granulocytes, and B cells). Therefore, the patients were closely monitored. The relative preservation of the peripheral circulating granulocytes, monocytes, and B lymphocytes suggests that this treatment does not adversely affect these cell populations. The use of CD4-positive and CD8-positive lymphocyte subsets as a prognostic indicator of HIV disease has been widely accepted. Intraindividual variations require multiple measurements. Fahey and colleagues (14) found that in patients with HIV-1 infection, the various ways of measuring CD4-positive T cells correlated well with one another. This was especially true of the correlation between the CD4:CD8 ratio and the percentage of CD4-positive cells in total lymphocytes, as the ratio was calculated from the percentages. Absolute numbers correlated less well, because the total and differential
Hypersensitivity Patient 1
Patient 3
Patient 2
Patient 4
Patient 5
-
-
-
-
+ (12 mm)t
-
-
-
-
-
-
+ (12 mm)
-
-
+ (14 mm)
+ (13 mm)
-
-
+ (18 mm) + (30 mm)
-
+ (13 mm) + (18 mm)
-
-
+ (13 mm)
+ (12 mm)
* Six months after first treatment. t Refers to millimeters of induration.
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273
leukocyte counts necessary to determine absolute numbers of cells vary widely (14). The relative stability of the percentage and absolute numbers of CD4-positive cells and the CD4:CD8 ratio during our study suggests that photopheresis does not decrease helper T lymphocytes in HIV-infected patients. In fact, reinfusion of extracorporeal^ treated autologous lymphocytes has apparently achieved encouraging results. Although the preliminary laboratory data appear promising, the small number of patients in our study limits the power of the statistical tests that can be applied and, therefore, the conclusions that can be drawn. The absence of a control group prevents comparison of the response of patients treated with photopheresis to a known standard with the same entrance criteria. Photopheresis sham control is considered inadequate for ethical reasons because it would expose patients to the risk for hypovolemic shock (4, 12), a side effect of photopheresis, with no apparent benefit. Although a group matched for findings could have been assigned to receive no treatment, this is not required by the United States Food and Drug Administration for a phase 1 trial. If the results for our patients were plotted against those of patients described in the literature, the course of the disease for four of the participants in our trial would differ from that of the previously described patients. For example, a representative patient in the study population of Masur and colleagues (15) had 17% CD4-positive cells with an absolute CD4-positive count of 310/mm3. Over a 14-month period, a precipitous drop occurred in both the percentage and the absolute number of CD4-positive cells, ultimately resulting in opportunistic infection. In contrast, of the five patients participating in our trial, two showed an increase in the absolute number of CD4-positive lymphocytes from baseline, with one remaining relatively stable after 15 months. The percentage of CD4-positive lymphocytes stabilized or increased in four of the five patients. In the same study, Masur and colleagues (15) found that patients with CD4-positive cell counts of less than 200 to 250/mm3 or less than 20% to 25% CD4-positive cells are at high risk for developing Pneumocystis carinn pneumonia and that those with CD4-positive cell counts of more than 200 to 250/mm3 or more than 20% to 25% CD4-positive cells can develop viral upper respiratory tract infections or serious pulmonary disease caused by common, community-acquired pathogens. In our study, although patients were exposed to both community- and hospital-borne pathogens and three of the five patients would have qualified for prophylactic therapy according to the criteria of Masur and colleagues (15), none developed P. carinii pneumonia or other opportunistic infections. This outcome suggests that extracorporeal photopheresis has not potentiated immunologic deterioration in this limited population. In another study population, Murray and colleagues (16) found that 77% to 88% of patients with the following conditions developed AIDS: skin-test anergy, less than 200 CD4-positive cells/mm3, and a CD4:CD8 ratio of less than 0.5. All five patients in our study had skin-test anergy, two of the five started had less than 274
200 CD4-positive cells/mm3, and one of the five had a CD4:CD8 ratio of less than 0.5. After treatment, however, four of the five patients regained delayed-type hypersensitivity; the two patients with CD4-positive cell levels of less than 200/mm3 showed increases after 6 months of therapy and their CD4:CD8 ratio rose to 0.5 or more. Patients with AIDS typically show poor primary and secondary humoral immune responses (17), but all five patients in our study showed increases in p24 and gpl20 antibodies. McDougal and colleagues (18) showed a correlation between antibody titer and the severity of immunodeficiency regardless of clinical status. Elevations of p24 and gpl20 antibodies, as in our study, were predictive of nonprogression to AIDS independent of their association with the levels of CD4-positive cells (18). Clinically, spontaneous involution of lymphadenopathy is known to occur with progression to AIDS and can be considered a poor prognostic sign. In contrast, the involution of lymph nodes in this study was not an isolated event but was accompanied by the maintenance of relative stability in the percentage and absolute numbers of CD4-positive cells during a period of approximately 15 months, stabilization of or decline in beta2microglobulin levels, an increase in antibody titers, and an inability to culture HIV in some patients. If the observed stabilization of or increase in the number of CD4-positive cells seen in the patients along with the increased antibody production is indeed an immune response to the virus, one might expect a suppression of viral replication, directly or indirectly related to the ultraviolet A-psoralen therapy. In-vitro studies have shown that an immune response can be engendered by exposing herpesvirus to psoralen and ultraviolet A. Redfield and colleagues (19) have shown that in-vitro exposure of herpesvirus to ultraviolet A light and psoralen caused the loss of infectivity in both the intact virus and virus-infected cells without the loss of viral antigenicity. In addition, treated virus-infected, antigen-bearing cells induced virus-specific lymphocyte proliferation in vitro (19). Such a directed response may be involved in the failure to culture HIV from peripheral blood mononuclear cells in three of our patients. Although it has been previously reported that HIV can be isolated more frequently from peripheral blood mononuclear cells of patients with early-stage infection than from those with AIDS (20, 21), Coombs and colleagues (22) recently isolated HIV from peripheral blood mononuclear cells in 97% of antibody-positive patients. The isolation technique used in our study has allowed isolation of HIV from peripheral blood mononuclear cells in 80% of antibody-positive patients (Michalski F. Personal communication). Although the mechanism of action of photopheresis still remains uncertain, it appears from the in-vitro and in-vivo studies by Edelson and colleagues (4) that specific T-cell clones are selectively targeted by photopheresis and then recognized by the immune system. These T-cell clones, although admixed with many smaller irrelevant clones, have been sufficiently altered by photopheresis to lead to an immunologic reaction against them after their reinfusion (13). Similar studies by Co-
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hen (23) in experimental autoimmune encephalomyelitis have shown that pathogenic T cells that have been altered by pressure could be presented to an animal in such a manner that rather than causing disease, they could actually prevent the development of the illness. Khavari and associates (24) obtained similar results using a photopheresis system. These investigators found that pathogenic T cells physically altered in vitro can be modified by photopheresis to prevent the development of experimental autoimmune encephalomyelitis in rodents. In addition, using whole blood, Edelson and colleagues (4) showed that a similar model could be effective for treating a coexisting progressive malignant disease without the in-vitro cloning required by the system used by Cohen (23). Photopheresis offers the significant advantage of a closed extracorporeal system for the possible inactivation of free and cell-associated virus. Reinfusion of the treated fraction into an immune system partially suppressed by the viral infection but not adversely affected by therapy may allow for the initiation of an immune response. We believe the current results are sufficiently promising to warrant an expanded clinical trial with a larger prospective group to evaluate fully this therapy. Acknowledgments: The authors thank Steven Armus, MD, postdoctoral fellow in infectious diseases and immunology, and the nursing staff at the Photopheresis Unit, Morristown Memorial Hospital, New Jersey; they also thank Frank Michalski, PhD, Metpath Laboratories, Teterboro, New Jersey, for assisting with HIV viral culture. Grant Support: In part by grants from Malcolm Forbes, the Amelior Foundation, the Whitehouse Fund, and the Reeve Foundation. Requests for Reprints: Emil Bisaccia, MD, Photopheresis Unit, Morristown Memorial Hospital, 100 Madison Avenue, Morristown, NJ 079621956. Current Author Addresses: Drs. Bisaccia and Klainer: Morristown Memorial Hospital, 100 Madison Avenue, Morristown, NJ 07962. Dr. Berger: Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. References 1. Masajo L, Rodighier G, Colombo J, Terlone V, Dallacqua F. Photo sensitizing furocoumarines: interactions with DNA and photoinactivation of DNA containing viruses. Experientia. 1965;21:22-4. 2. Nakashima K, Shatkin AJ. Photochemical cross-linking of retrovirus genome RNA in situ and inactivation of viral transcriptase. J Biol Chem. 1978;253:8680-2. 3. Quinnan GV Jr, Wells MA, Wittek AE, et al. Inactivation of human T-cell virus, type III by heat, chemicals and irradiation. Transfusion. 1986;26:481-3. 4. Edelson R, Berger C, Gasparro F, et al. Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results. N Engl J Med. 1987;316:297-303.
5. Gallo RC. HIV—the cause of AIDS: an overview of its biology, mechanisms of disease induction and our attempts to control it. J Acquire Immune Defic Syndr. 1988;1:521-35. 6. Rothenberg R, Woelfel M, Stoneburner R, Milberg J, Parker R, Truman B. Survival with the acquired immunodeficiency syndrome. Experience with 5833 cases in New York City. N Engl J Med. 1987;317:1297-302. 7. Redfield RR, Wright DC, Tramont EC. The Walter Reed staging classification for HTLV-III/LAV infection. N Engl J Med. 1986;314: 131-2. 8. Kung PC, Berger CL, Goldstein G, LoGerfo P, Edelson RL. Cutaneous T-cell lymphoma: characterization by monoclonal antibodies. Blood. 1981;57:261-6. 9. Lacey JN, Forbes MA, Waugh MA, Cooper EH, Hambling MH. Serum beta 2 -microglobulin and human immunodeficiency virus infection. Aids. 1987;1:123-7. 10. Velleca WN, Palmer DF, Feorno RW, et al. Training Manual of the Center for Disease Control: Isolation Culture and Identification of Human T-cell Lymphotropic Virus HI, Lymphadenopathy Associated Virus. Atlanta, Georgia: Centers for Disease Control; 1986. 11. Jackson GG, Paul DA, Falk LA, et al. Human immunodeficiency virus (HIV) antigenemia (p24) in acquired immune deficiency syndrome (AIDS) and the effect of treatment with zidovudine (AZT). Ann Intern Med. 1988;108:175-80. 12. Ng VL, Chiang CS, Debouck C, McGrath MS, Grove TH, Mills J. Reliable confirmation of antibodies to human immunodeficiency virus type I (HIV-1) with enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, sol, and eng genes. J Clin Microbiol. 1989;27:977-82. 13. Edelson RL. Light-activated drugs. Sci Am. 1988;259:68-75. 14. Fahey JL, Taylor JM, Detels R, et al. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus, type 1. N Engl J Med. 1990;322:166-72. 15. Masur H, Ognibene FP, Yarchoan R, et al. CD4 counts as predictors of opportunistic pneumonias in human immunodeficiency virus (HIV) infection. Ann Intern Med. 1989;111:223-31. 16. Murray HW, Godbold JH, Jurica KB, Roberts RB. Progression to AIDS in patients with lymphadenopathy or AIDS-related complex: reappraisal of risk and predictive factors. Am J Med. 1989;86:533-8. 17. Ammann AJ, Schiffman G, Abrams D, Volberding P, Ziegler J, Conant M. B-cell immunodeficiency in acquired immune deficiency syndrome. JAMA. 1984;251:1447-9. 18. McDougal JS, Kennedy MS, Nicholson JK, et al. Antibody response to human immunodeficiency virus in homosexual men. Relation of antibody specificity, titer, and isotype to clinical status, severity of immunodeficiency, and disease progression. J Clin Invest. 1987;80: 316-24. 19. Redfield DC, Richman DD, Oxman MN, Kronenberg LH. Psoralen inactivation of influenza and herpes simplex viruses and of virusinfected cells. Infect Immun. 1981;32:1216-26. 20. Levy JA, Shimabukuro J. Recovery of AIDS-associated retroviruses from patients with AIDS or AIDS-related conditions and from clinically healthy individuals. J Infect Dis. 1985;152:734-8. 21. Andrews CA, Sullivan JL, Brettler DB, et al. Isolation of human immunodeficiency virus from hemophiliacs: correlation with clinical symptoms and immunologic abnormalities. J Pediatr. 1987;111:6727. 22. Coombs RW, Collier AC, Allain JP, et al. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989;321:1626-31. 23. Cohen IR. Autoimmunity: physiologic and pernicious. Adv Intern Med. 1984;29:147-65. 24. Khavari PA, Edelson RL, Lider O, Gasparro FP, Weiner HL, Cohen IR. Specific vaccination against photoinactivated cloned T-cells. Clin Res [Abstract]. 1988;36:662A.
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Objective: To determine side effects of extracorporeal photopheresis in the treatment of patients with the acquired immunodeficiency syndrome (AIDS)-related complex, and to gain early evidence of efficacy of the treatment. Design: Uncontrolled trial. Setting: Tertiary referral center. Patients: Five patients with AIDS-related complex: three homosexual men and one man and one woman with histories of intravenous drug abuse. One patient, a homosexual man, withdrew from therapy after 5 months but returned for monthly clinical and laboratory evaluations. Intervention: Monthly treatments with extracorporeal photopheresis. Measurements and Main Results: Symptoms resolved in four patients. Lymphadenopathy disappeared in all five. Four patients had delayed-hypersensitivity reactions to skin testing (as defined by the Walter Reed staging classification). All showed increases in p24 and gpl20 antibody levels. The CD4-cell percentage increased in four patients and declined in one after 6 months of therapy, but the absolute CD4 count decreased in two patients. At 15 months, the CD4 percentage remained at or increased over the baseline value in three patients still in the study but decreased in one. Levels of Beta2-microglobulin decreased or remained stable in four patients. All patients were culture positive for the human immunodeficiency virus (HIV) before treatment. One patient had a negative viral culture after 5 months of treatment with confirmation. Two other patients became HIV culture negative, one at 14 and one at 15 months: The former patient became positive at 15 months and the latter patient remained negative at 16 months. Conclusions: The preliminary results suggest that extracorporeal photopheresis deserves further evaluation as therapy for AIDSrelated complex.
Annals of Internal Medicine. 1990;113:270-275. From Morristown Memorial Hospital, Morristown, New Jersey, and Columbia University College of Physicians and Surgeons, New York, New York. For current author addresses, see end of text. 270
© 1990 American College of Physicians
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I n vitro, virus-inactivating agents may function by modifying viral coats or damaging viral nucleic acid, or by doing both simultaneously. Photochemical inactivation of DNA viruses using psoralen derivatives was first demonstrated in vitro in 1961; the technique was later extended to RNA viruses. Irradiation of viruses with long-wavelength ultraviolet A in the presence of psoralen results in cross-linking of base-pair regions of nucleic acids (1, 2), with formation of covalent monoadducts. Both DNA and RNA viruses, including the human immunodeficiency virus (HIV), have been inactivated in vitro by psoralen photoreaction (3). Recently, in a study by Edelson and associates (4), patients with cutaneous T-cell lymphoma, a CD4-positive lymphocytic malignancy, showed a marked clinical response to photopheresis (4). In this therapy, 8-methoxypsoralen is administered orally before ultraviolet A irradiation of patients' leukocytes in an extracorporeal photopheresis device; after irradiation, cells are reinfused. The primary target for HIV infection is the CD4positive helper-inducer T-cell lymphocyte; the cytopathic consequences of the infection are most prominent in this lymphocytic cell population. Additional susceptible cell types include those of the macrophagemonocyte lineage. Human immunodeficiency virus infection of these cells in the peripheral blood, lymphatics, and other tissues has been well documented. The mechanism underlying the CD4 treated-cell depletion that occurs in vivo in HIV infection may involve cell fusion, indirect and undefined cytopathic processes, and possibly an autoimmune mechanism (5). Such depletion causes immunologic debilitation, which is ultimately fatal within 12 to 48 months as these patients progress from AIDS-related complex to AIDS. The terminal event is most commonly an infection (6), as is the case in patients with cutaneous T-cell lymphoma (4). The accessibility of the blood leukocytes and, therefore, the CD4-positive cells to an extracorporeal system involving psoralen and ultraviolet A light led to the consideration of a phase 1 trial in patients with HIV infection. Because no adequate animal model is available for studying HIV infection in vivo or for evaluating the response to new potential therapies, a trial was undertaken in humans. A pilot study was begun with five volunteers who had photopheresis on 2 consecutive days at monthly intervals. The results of the initial 6 months of phase 1 treatment are reported for five patients with AIDS-related complex; the study, having extended to 14 to 15 months, is now a phase 2 trial. A phase 1 study is designed to determine side effects and, if possible, to gain early evidence of effectiveness. A phase 2 trial is done to obtain a preliminary eval-
Table 1. Clinical Characteristics Characteristic Sex, age, y Predisposing factor Walter Reed staging classification Known duration of HIV antibody positivity, y Weight, kg Before treatment After treatment* Presentt Absolute lymphocyte count, /mm3 Before treatment After treatment Present CD4-positive cells, /mm3 (%) Before treatment After treatment Present CD4:CD8 ratio Before treatment After 6 months Present p24 antibody level (absorbance)§ Before treatment After 6 months Present gpl20 antibody level (absorbance)|| Before treatment After 6 months Present Virus culturell Before treatment After 6 months Present Beta2-microglobulin, mg/L Before treatment After treatment Present p24 antigen, pglmL Before treatment After treatment Present
and Laboratory
Results
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
M, 38
M, 30
M, 27
Homosexuality 4
Homosexuality 5
Homosexuality 5
M, 42 Intravenous drug abuse 4
F, 27 Intravenous drug abuse 5
3
2
4
2
1
71.3 70.5 71.0
64.5 66.0 68.7
63.6 64.7 64.5
2600 1768 1620
1824 1533 1554
1617 893 546
109 108 110 1024 2067 2176
61.0 57.7 56.0 1936 522 594
104 (4)t 601 (34) 181 (25)
529 (29) 475 (31) 451 (29)
550 (34) 330 (40) 282 (52)
113 (11) 682 (33) 587 (27)
232 (12) 68 (13) 42(7)
0.2 0.6 0.5
0.8 0.7 0.7
1.0 1.1 1.1
0.8 0.8 0.8
0.1 0.8 0.8
2.52 2.76 2.78
0.12 0.74 0.79
0.72 1.69 2.02
0.62 1.76 2.50
1.41 2.89 2.68
2.22 1.68 2.89
0.03 0.14 2.59
1.04 2.07 2.46
0.21 1.25 2.54
0.46 1.95 2.63
+ + +
+
+
+ +
-
+ + -
+ + +
NA 2.8 2.6
3.6 3.6 3.6
NA 4.3 3.1
3.9 4.3 NA
NA 495 258
NA
NA 135 54
NA
NA** 7.6 6.2 NA
-
-
-
* After 6 months of therapy except in Patient 3 who received only 5 months of therapy (see text). t After 14 to 15 months of therapy (see text). X Percentage of peripheral T cells that are CD4 positive. § Standard deviation for p24 antibody (± 13%). |j Standard deviation for gpl20 antibody (± 20%). 11 Culture was done using peripheral blood mononuclear cells. ** NA = not available.
uation of the efficacy for a particular indication. The preliminary results suggest that serious short-term toxicity is not an inherent feature of the photopheresis regimen. Four of the five patients had an encouraging response. Patients and Methods Five patients with AIDS-related complex were selected from the six patients who volunteered for the study. The patient who was not selected was disqualified because he was enrolled in another drug trial. All five patients had enlarged lymph nodes (1 cm or more) at two or more sites; and unexplained systemic symptoms, including intermittent or continuous fever for more than 1 month, repeated night sweats, a debilitating, persistent weight loss of at least 5%, and a decreased percentage of CD4-positive cells. The study was done according to the provisions for the treatment of human subjects of the Institu-
tional Review Board at Morristown Memorial Hospital, Morristown, New Jersey. The study group comprised three homosexual men who ranged in age from 27 to 39 years, one 42-year-old man with a history of intravenous drug abuse, and one 27-year-old woman with a history of intravenous drug abuse. One to four years had elapsed between the patients' first known HIV antibody positivity and their enrollment in the study (Table 1). Only patients who had not had any previous therapy for HIV infection were enrolled. The patients were interviewed by the principal investigators and answered a quality-of-life questionnaire (available from the authors on request). On the basis of the Walter Reed (WR) staging classification (7), patients ranged from WR4 to WR5 (Table 1). Cervical, supraclavicular, axillary, epitrochlear, and inguinal lymph nodes were palpated before enrollment and before each treatment by an experienced physician. Lymph node enlargement was defined as a palpable node of 1 cm or more at two
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Table 2. Lymph Node Examination Patient
Cervical Right Left
before
Treatment
Supraclavicular Right Left
Axillary Right Left
cm
1 2
2 1
3 1
... ...
... ...
1 1
3 4 5
... 1 1
... 1 1
... ... ...
... ... ...
... ... ...
or more sites. The size of the lymph node was approximated by the use of standard skin calipers (Table 2). Nonblinded skin testing with four standard recall antigens was done in the standard manner to induce a delayed-hypersensitivity reaction. A cutaneous response was considered positive if skin induration of at least 5 mm resulted from application of two or more of the following four test antigens: Trichophyton, mumps, Candida, and intermediate-strength tuberculin purified protein derivative (Table 3). Skin biopsy with histologic confirmation was obtained when indicated and was evaluated by a blinded observer. Pretreatment studies included an enzyme-linked immunosorbent assay (ELISA) for HIV and a confirmatory Western blot, antibody tests (HIVAGEN-SKF, SmithKline Beecham, West Norriton, Pennsylvania), HIV isolation by p24 antigen capture (Metpath, Teterboro, New Jersey), peripheral blood phenotyping, hepatitis profile, VDRL test, antinuclear antibody test, chest roentgenogram, and electrocardiogram. Pretreatment and monthly post-treatment studies included a complete blood count, platelet count, erythrocyte sedimentation rate, reticulocyte count, a 20-parameter automated analysis, and urinalysis. In month 3 of the trial, determinations of cytomegalovirus and Epstein-Barr virus titers, immunoglobulin electropheresis, and assessments of beta2-microglobulin and p24 antigen levels were added. Isolation of HIV was repeated every 3 months. All patients were weighed before enrollment and before each monthly treatment. Skin testing to the recall antigens of tuberculin, mumps, Candida, and Trichophyton was done before treatment and at the completion of approximately 6 months of therapy. Lymphocytes isolated from peripheral blood using FicollHypaque separation were stained with monoclonal antibodies, and the percentages of CD3-positive, CD4-positive, and CD8positive T cells were determined by flow cytometry (8). The absolute numbers of CD4-positive and CD8-positive T cells were calculated from the total and differential leukocyte counts. We measured beta2-microglobulin by radioimmunoassay (Pharmacia, Uppsala, Sweden) using standards provided by the manufacturer (9). Isolation of virus was done using a modification of the technique developed by the Centers for Disease Control (10). Lymphocytes obtained from heparin-treated whole blood were isolated by Ficoll-Hypaque separation, cultured, and assayed for p24 antigen at 7, 14, 21, and 28 days. Results of virus isolation were confirmed at 3-month intervals. We measured HIV p24 antigen using a solid-phase, sandwich-type enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, Illinois) (11). This assay uses a polyclonal HIV antibody and a purified viral lysate as a standard; therefore, p24 antigen and other viral proteins are measured. An assay was considered positive if the optical density of the culture supernatant was greater than that of the standard. The specificity of each positive specimen was confirmed by a neutralizing assay using 50 /ig of a polyclonal human anti-HIV IgG solution. A reduction of more than 50% of the recorded absorbance was considered to be confirmatory (11). We tested for HIV antibodies using the Western blot and HIVAGEN assays. The HIVAGEN assay is based on six purified recombinant antigens and is used to test for HIV antibodies in the screened sera. It has been suggested that this assay be used for longitudinal evaluation of seropositive patients for disease progression or antiviral therapy (11). 272
Epitrochlear Right Left
1 2
... ...
1.75 ... ...
1 ... ...
1
2 ... ...
3 3
3
2 3 3
Western blot assays were done according to the instructions of the manufacturer (Du Pont, Wilmington, Delaware). In our study, photopheresis was done using the UVAR instrument (Therakos, West Chester, Pennsylvania), which integrates an initial discontinuous leukapheresis step with exposure to ultraviolet light in a single apparatus. The procedure involved pooling 240 mL of leukocyte-enriched blood with 300 mL of the patient's plasma (removed 2 hours after ingestion of 0.4 to 0.6 mg/kg body weight of 8-methoxypsoralen) and 200 mL of sterile normal saline. The leukocyte-enriched fraction was obtained by centrifugation, using a 125-mL pediatric bowl and a six-cycle leukapheresis technique (UVAR instrument, Therakos, West Chester, Pennsylvania). The total volume was then passed as a 1-mm film through a sterile irradiation chamber to expose it to ultraviolet A energy delivered by a fluorescent source. After exposure of blood to ultraviolet A, the entire amount was returned to the patient (4). Each patient received a monthly treatment. Results Four patients received a total of 12 treatments each as part of the 6-month phase 1 study that has now continued through 14 to 15 months, evolving into a phase 2 trial. The fifth patient (Patient 3) voluntarily withdrew from the phase 1 study after 5 months of treatment (10 treatments) but continues to return for monthly clinical follow-up and laboratory evaluation. All patients had a positive Western blot, a positive test for antibody to two HIV-1 gene products, and a positive HIV culture. On the basis of the physical examination, clinical indices, monthly antibody tests, p24 antigen studies, and the percentage or absolute number of CD4-positive cells, four of five patients were judged to have had either a stabilization of their disease or an encouraging response to treatment (Table 1). The absence of significant toxicity during the treatment course was suggested by the stabilization of the percentage of CD4-positive cells, the CD4:CD8 ratio, or the absolute number of CD4-positive cells, and an increase in antibody titers (Table 1). On the basis of responses to the quality-of-life questionnaire and a review of symptoms in conjunction with evaluation of clinical status, four patients showed some improvement. Initially, the increase in energy lasted for approximately 1 week after treatment. By the third treatment, the increase in energy persisted, and the general quality of life improved. Most notably, the first patient treated, a homosexual man who before treatment was able to walk up a flight of stairs only with difficulty, is now capable of jogging 3.5 miles a day and lifting weights. In addition, two other participants have shown similar subjective improvement: Both are now attending college and working full-time. The one patient who did not show a persistent rise in energy level was
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3 2
... ...
study have shown either a stabilization of or an increase from pretreatment levels in the percentage and absolute numbers of CD4-positive cells and in the CD4.CD8 ratio. In these same three patients, the absolute lymphocyte count increased in one, was relatively stable in one, and decreased in one. The beta 2 -microglobulin levels decreased in two patients and remained relatively stable in the other (Table 1). The p24 and gpl20 antibody levels increased in all patients (Table 1).
the woman with a history of intravenous drug abuse who had three small children. Appetite increased and weight remained stable in all patients except this woman, whose weight decreased 3.3 kg during the 6month treatment period (Table 1). Fever and night sweats disappeared after 3 months of therapy. Lymphadenopathy (Table 2) disappeared in all patients by the third month of therapy and remained absent. Three patients were anergic in skin tests before treatment; two of these patients reacted positively to two skin-test antigens after therapy. The two remaining patients reacted to a single antigen before therapy and both responded to a second antigen after therapy, thereby meeting our case definition for positive skin-test reactivity (Table 3). In two patients, the positive skintest sites were biopsied, and delayed-hypersensitivity reactions were confirmed by a blinded observer. During the 15-month period of treatment, common communityacquired upper respiratory tract infections were managed in all patients without difficulty. Except for normal variations, the hemoglobin, hematocrit, reticulocyte count, and platelet count remained stable. There were minor variations in the leukocyte count. In all patients, erythrocyte sedimentation rate, results of urinalysis, and results of a 20-parameter automated analysis remained unchanged, except for minor elevations in aspartate aminotransferase and alanine aminotransferase. Epstein-Barr virus titers remained constant in all but one patient: The titer of his IgG capsid antigen decreased from 1:1280 to 1:160, which paralleled an increase in HIV antibody titers. Cytomegalovirus titers remained unchanged in all. At 6 months, those patients who were p24 antigen negative before treatment remained negative, and those who were positive remained positive with no significant changes in titer. By 15 months, the p24 antigen levels had decreased in the two antigen-positive patients (Table 1). Cultures of blood for HIV became negative in Patient 3 after the fifth treatment; this was confirmed twice during the ensuing 9 months. Two additional patients, Patients 1 and 4, became negative for HIV at 14 and 15 months, respectively. Patient 4 was still negative at 16 months; Patient 1, however, became positive at 15 months. During approximately 15 months of treatment, the three male patients who remained in the Table 3. Skin Testing for Delayed Test Trichophyton Before treatment After treatment* Tuberculin purified protein derivative (intermediate strength) Before treatment After treatment Mumps Before treatment After treatment Candida Before treatment After treatment
Discussion The paramount concern with the application of photopheresis in patients with HIV infection is that the reinfusion of T cells treated or activated by photopheresis would lead to further depletion of the CD4-positive population. Data from in-vitro studies have shown that a substantial loss of viability occurs over time in normal lymphocytes on exposure to 8-methoxypsoralen and ultraviolet A light (12). Approximately one third of the circulating lymphocytes are treated in the described methods, and the possible reduction of lymphocyte viability in patients with HIV infection might have resulted in severe harm to the patients. Because psoralen enters all nucleated cells, DNA adduct formation occurs in every cell exposed to the drug and ultraviolet A light. This cross-linking of DNA is not repairable by the cells' normal mechanisms and has potential implications for other cells of the immune system (for example, monocytes, granulocytes, and B cells). Therefore, the patients were closely monitored. The relative preservation of the peripheral circulating granulocytes, monocytes, and B lymphocytes suggests that this treatment does not adversely affect these cell populations. The use of CD4-positive and CD8-positive lymphocyte subsets as a prognostic indicator of HIV disease has been widely accepted. Intraindividual variations require multiple measurements. Fahey and colleagues (14) found that in patients with HIV-1 infection, the various ways of measuring CD4-positive T cells correlated well with one another. This was especially true of the correlation between the CD4:CD8 ratio and the percentage of CD4-positive cells in total lymphocytes, as the ratio was calculated from the percentages. Absolute numbers correlated less well, because the total and differential
Hypersensitivity Patient 1
Patient 3
Patient 2
Patient 4
Patient 5
-
-
-
-
+ (12 mm)t
-
-
-
-
-
-
+ (12 mm)
-
-
+ (14 mm)
+ (13 mm)
-
-
+ (18 mm) + (30 mm)
-
+ (13 mm) + (18 mm)
-
-
+ (13 mm)
+ (12 mm)
* Six months after first treatment. t Refers to millimeters of induration.
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273
leukocyte counts necessary to determine absolute numbers of cells vary widely (14). The relative stability of the percentage and absolute numbers of CD4-positive cells and the CD4:CD8 ratio during our study suggests that photopheresis does not decrease helper T lymphocytes in HIV-infected patients. In fact, reinfusion of extracorporeal^ treated autologous lymphocytes has apparently achieved encouraging results. Although the preliminary laboratory data appear promising, the small number of patients in our study limits the power of the statistical tests that can be applied and, therefore, the conclusions that can be drawn. The absence of a control group prevents comparison of the response of patients treated with photopheresis to a known standard with the same entrance criteria. Photopheresis sham control is considered inadequate for ethical reasons because it would expose patients to the risk for hypovolemic shock (4, 12), a side effect of photopheresis, with no apparent benefit. Although a group matched for findings could have been assigned to receive no treatment, this is not required by the United States Food and Drug Administration for a phase 1 trial. If the results for our patients were plotted against those of patients described in the literature, the course of the disease for four of the participants in our trial would differ from that of the previously described patients. For example, a representative patient in the study population of Masur and colleagues (15) had 17% CD4-positive cells with an absolute CD4-positive count of 310/mm3. Over a 14-month period, a precipitous drop occurred in both the percentage and the absolute number of CD4-positive cells, ultimately resulting in opportunistic infection. In contrast, of the five patients participating in our trial, two showed an increase in the absolute number of CD4-positive lymphocytes from baseline, with one remaining relatively stable after 15 months. The percentage of CD4-positive lymphocytes stabilized or increased in four of the five patients. In the same study, Masur and colleagues (15) found that patients with CD4-positive cell counts of less than 200 to 250/mm3 or less than 20% to 25% CD4-positive cells are at high risk for developing Pneumocystis carinn pneumonia and that those with CD4-positive cell counts of more than 200 to 250/mm3 or more than 20% to 25% CD4-positive cells can develop viral upper respiratory tract infections or serious pulmonary disease caused by common, community-acquired pathogens. In our study, although patients were exposed to both community- and hospital-borne pathogens and three of the five patients would have qualified for prophylactic therapy according to the criteria of Masur and colleagues (15), none developed P. carinii pneumonia or other opportunistic infections. This outcome suggests that extracorporeal photopheresis has not potentiated immunologic deterioration in this limited population. In another study population, Murray and colleagues (16) found that 77% to 88% of patients with the following conditions developed AIDS: skin-test anergy, less than 200 CD4-positive cells/mm3, and a CD4:CD8 ratio of less than 0.5. All five patients in our study had skin-test anergy, two of the five started had less than 274
200 CD4-positive cells/mm3, and one of the five had a CD4:CD8 ratio of less than 0.5. After treatment, however, four of the five patients regained delayed-type hypersensitivity; the two patients with CD4-positive cell levels of less than 200/mm3 showed increases after 6 months of therapy and their CD4:CD8 ratio rose to 0.5 or more. Patients with AIDS typically show poor primary and secondary humoral immune responses (17), but all five patients in our study showed increases in p24 and gpl20 antibodies. McDougal and colleagues (18) showed a correlation between antibody titer and the severity of immunodeficiency regardless of clinical status. Elevations of p24 and gpl20 antibodies, as in our study, were predictive of nonprogression to AIDS independent of their association with the levels of CD4-positive cells (18). Clinically, spontaneous involution of lymphadenopathy is known to occur with progression to AIDS and can be considered a poor prognostic sign. In contrast, the involution of lymph nodes in this study was not an isolated event but was accompanied by the maintenance of relative stability in the percentage and absolute numbers of CD4-positive cells during a period of approximately 15 months, stabilization of or decline in beta2microglobulin levels, an increase in antibody titers, and an inability to culture HIV in some patients. If the observed stabilization of or increase in the number of CD4-positive cells seen in the patients along with the increased antibody production is indeed an immune response to the virus, one might expect a suppression of viral replication, directly or indirectly related to the ultraviolet A-psoralen therapy. In-vitro studies have shown that an immune response can be engendered by exposing herpesvirus to psoralen and ultraviolet A. Redfield and colleagues (19) have shown that in-vitro exposure of herpesvirus to ultraviolet A light and psoralen caused the loss of infectivity in both the intact virus and virus-infected cells without the loss of viral antigenicity. In addition, treated virus-infected, antigen-bearing cells induced virus-specific lymphocyte proliferation in vitro (19). Such a directed response may be involved in the failure to culture HIV from peripheral blood mononuclear cells in three of our patients. Although it has been previously reported that HIV can be isolated more frequently from peripheral blood mononuclear cells of patients with early-stage infection than from those with AIDS (20, 21), Coombs and colleagues (22) recently isolated HIV from peripheral blood mononuclear cells in 97% of antibody-positive patients. The isolation technique used in our study has allowed isolation of HIV from peripheral blood mononuclear cells in 80% of antibody-positive patients (Michalski F. Personal communication). Although the mechanism of action of photopheresis still remains uncertain, it appears from the in-vitro and in-vivo studies by Edelson and colleagues (4) that specific T-cell clones are selectively targeted by photopheresis and then recognized by the immune system. These T-cell clones, although admixed with many smaller irrelevant clones, have been sufficiently altered by photopheresis to lead to an immunologic reaction against them after their reinfusion (13). Similar studies by Co-
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hen (23) in experimental autoimmune encephalomyelitis have shown that pathogenic T cells that have been altered by pressure could be presented to an animal in such a manner that rather than causing disease, they could actually prevent the development of the illness. Khavari and associates (24) obtained similar results using a photopheresis system. These investigators found that pathogenic T cells physically altered in vitro can be modified by photopheresis to prevent the development of experimental autoimmune encephalomyelitis in rodents. In addition, using whole blood, Edelson and colleagues (4) showed that a similar model could be effective for treating a coexisting progressive malignant disease without the in-vitro cloning required by the system used by Cohen (23). Photopheresis offers the significant advantage of a closed extracorporeal system for the possible inactivation of free and cell-associated virus. Reinfusion of the treated fraction into an immune system partially suppressed by the viral infection but not adversely affected by therapy may allow for the initiation of an immune response. We believe the current results are sufficiently promising to warrant an expanded clinical trial with a larger prospective group to evaluate fully this therapy. Acknowledgments: The authors thank Steven Armus, MD, postdoctoral fellow in infectious diseases and immunology, and the nursing staff at the Photopheresis Unit, Morristown Memorial Hospital, New Jersey; they also thank Frank Michalski, PhD, Metpath Laboratories, Teterboro, New Jersey, for assisting with HIV viral culture. Grant Support: In part by grants from Malcolm Forbes, the Amelior Foundation, the Whitehouse Fund, and the Reeve Foundation. Requests for Reprints: Emil Bisaccia, MD, Photopheresis Unit, Morristown Memorial Hospital, 100 Madison Avenue, Morristown, NJ 079621956. Current Author Addresses: Drs. Bisaccia and Klainer: Morristown Memorial Hospital, 100 Madison Avenue, Morristown, NJ 07962. Dr. Berger: Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. References 1. Masajo L, Rodighier G, Colombo J, Terlone V, Dallacqua F. Photo sensitizing furocoumarines: interactions with DNA and photoinactivation of DNA containing viruses. Experientia. 1965;21:22-4. 2. Nakashima K, Shatkin AJ. Photochemical cross-linking of retrovirus genome RNA in situ and inactivation of viral transcriptase. J Biol Chem. 1978;253:8680-2. 3. Quinnan GV Jr, Wells MA, Wittek AE, et al. Inactivation of human T-cell virus, type III by heat, chemicals and irradiation. Transfusion. 1986;26:481-3. 4. Edelson R, Berger C, Gasparro F, et al. Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results. N Engl J Med. 1987;316:297-303.
5. Gallo RC. HIV—the cause of AIDS: an overview of its biology, mechanisms of disease induction and our attempts to control it. J Acquire Immune Defic Syndr. 1988;1:521-35. 6. Rothenberg R, Woelfel M, Stoneburner R, Milberg J, Parker R, Truman B. Survival with the acquired immunodeficiency syndrome. Experience with 5833 cases in New York City. N Engl J Med. 1987;317:1297-302. 7. Redfield RR, Wright DC, Tramont EC. The Walter Reed staging classification for HTLV-III/LAV infection. N Engl J Med. 1986;314: 131-2. 8. Kung PC, Berger CL, Goldstein G, LoGerfo P, Edelson RL. Cutaneous T-cell lymphoma: characterization by monoclonal antibodies. Blood. 1981;57:261-6. 9. Lacey JN, Forbes MA, Waugh MA, Cooper EH, Hambling MH. Serum beta 2 -microglobulin and human immunodeficiency virus infection. Aids. 1987;1:123-7. 10. Velleca WN, Palmer DF, Feorno RW, et al. Training Manual of the Center for Disease Control: Isolation Culture and Identification of Human T-cell Lymphotropic Virus HI, Lymphadenopathy Associated Virus. Atlanta, Georgia: Centers for Disease Control; 1986. 11. Jackson GG, Paul DA, Falk LA, et al. Human immunodeficiency virus (HIV) antigenemia (p24) in acquired immune deficiency syndrome (AIDS) and the effect of treatment with zidovudine (AZT). Ann Intern Med. 1988;108:175-80. 12. Ng VL, Chiang CS, Debouck C, McGrath MS, Grove TH, Mills J. Reliable confirmation of antibodies to human immunodeficiency virus type I (HIV-1) with enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, sol, and eng genes. J Clin Microbiol. 1989;27:977-82. 13. Edelson RL. Light-activated drugs. Sci Am. 1988;259:68-75. 14. Fahey JL, Taylor JM, Detels R, et al. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus, type 1. N Engl J Med. 1990;322:166-72. 15. Masur H, Ognibene FP, Yarchoan R, et al. CD4 counts as predictors of opportunistic pneumonias in human immunodeficiency virus (HIV) infection. Ann Intern Med. 1989;111:223-31. 16. Murray HW, Godbold JH, Jurica KB, Roberts RB. Progression to AIDS in patients with lymphadenopathy or AIDS-related complex: reappraisal of risk and predictive factors. Am J Med. 1989;86:533-8. 17. Ammann AJ, Schiffman G, Abrams D, Volberding P, Ziegler J, Conant M. B-cell immunodeficiency in acquired immune deficiency syndrome. JAMA. 1984;251:1447-9. 18. McDougal JS, Kennedy MS, Nicholson JK, et al. Antibody response to human immunodeficiency virus in homosexual men. Relation of antibody specificity, titer, and isotype to clinical status, severity of immunodeficiency, and disease progression. J Clin Invest. 1987;80: 316-24. 19. Redfield DC, Richman DD, Oxman MN, Kronenberg LH. Psoralen inactivation of influenza and herpes simplex viruses and of virusinfected cells. Infect Immun. 1981;32:1216-26. 20. Levy JA, Shimabukuro J. Recovery of AIDS-associated retroviruses from patients with AIDS or AIDS-related conditions and from clinically healthy individuals. J Infect Dis. 1985;152:734-8. 21. Andrews CA, Sullivan JL, Brettler DB, et al. Isolation of human immunodeficiency virus from hemophiliacs: correlation with clinical symptoms and immunologic abnormalities. J Pediatr. 1987;111:6727. 22. Coombs RW, Collier AC, Allain JP, et al. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989;321:1626-31. 23. Cohen IR. Autoimmunity: physiologic and pernicious. Adv Intern Med. 1984;29:147-65. 24. Khavari PA, Edelson RL, Lider O, Gasparro FP, Weiner HL, Cohen IR. Specific vaccination against photoinactivated cloned T-cells. Clin Res [Abstract]. 1988;36:662A.
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