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[CANCER RESEARCH 44, 5894-5901, December 1984] Immunological Characterizations of Patients with Acquired Immune Deficiency Syndrome, Acquired Immune Deficiency Syndrome-related Symptom Complex, and a Related Life-Style1 Evan M. Hersh,2 P. W. A. Mansell, J. M. Reuben, A. Rios, and G. R. Newell Departments of Clinical Immunology and Biological Therapy [E. M. H., P. W. A. M., J. M. R., A. R.], Laboratory Medicine [E. M. H., J. M. R.], and Cancer Prevention [P. W. A. M., A. R., G. R. N.], The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 ABSTRACT Immunological, hematological, and biochemical studies were done at the time of referral in 135 homosexual subjects, 28 of whom were symptom free (SF), 74 of whom had the acquired immune deficiency syndrome (AIDS)-related symptom complex (ARC), and 33 of whom had AIDS with Kaposi's sarcoma, opportunistic infection, or both. Of 38 laboratory parameters, 11 were significantly different than controls in the SF patients, 19 in the ARC patients, and 20 in the AIDS patients. In SF patients, delayed hypersensitivity was significantly suppressed for 6 of 12 recall antigens. In addition, the percentage of circulating lympho cytes, the percentage of T3+ cells, the percentage and absolute number of T4+ cells, the T4/T8 ratio, the blastogenic responses to phytohemagglutinin, pokeweed mitogen, and concanavalin A were depressed significantly in this group. In contrast, the per centage and absolute granulocyte count, the serum lysozyme, and the serum thymosin <*i were significantly elevated in these patients. In patients with more advanced disease (ARC and AIDS), immunological and hematological parameters tended to worsen. Thus, in the AIDS patients the white blood cell count, percentage, and absolute T11+ cells, absolute T3+ cells, per centage of T4+ cells and absolute level of B-cells, as well as the monocyte adherence and delayed hypersensitivity responses to 12 of 12 recall antigens were depressed. Serum levels of thy mosin «! were equally elevated in all three groups. Serum Interferon was found in 15 of 18 opportunistic infection patients with or without Kaposi's sarcoma, in 3 of 9 Kaposi's sarcoma patients without opportunistic infection, but in none of the ARC or SF patients. This study has demonstrated that SF sexually active homosexuals have a characteristic pattern of immune deficiency and that immunodeficiency worsens as one compares SF to ARC to AIDS patients. The study has provided a data base for the development of prognostic criteria and for charac terization and evaluation of immunorestorative and immunomodulatory therapy. since its initial identification in 1979 (1). Initially detected in homosexuals in New York City and California, it has now been identified in at least 5 other high-risk groups (32), is occasionally seen in subjects without a known predisposing life-style factor, and it has spread to countries in both Europe (11) and the Far East. There is no known treatment for the underlying immuno deficiency, and it is associated with a mortality of at least 40% and perhaps as high as 80% or more (10, 27). AIDS is a clinical syndrome characterized by major Ol or cancer, mainly KS, or both, associated with at least 2 defined immune abnormalities in patients without a known cause for the immunodeficiency. Known causes of immunodeficiency which exclude patients from diagnosis of AIDS include ¡mmunosuppressive drug therapy, preexisting cancer or other known dis eases with secondary immunodeficiency, and known congenital or acquired immunodeficiency diseases. Groups in which AIDS has been observed to occur include homosexuals (13), Haitian immigrants (2), hemophiliacs (3), i.v. drug abusers (12), prisoners (14), offspring of one of the above (31), and heterosexual partners of one of the above (28). Others may be discovered, since about 5% of cases have no recognized predisposing factor. A complex of symptoms (fever, night sweats, lymphadenopathy, weight loss, diarrhea, and fatigue) usually precedes the development of AIDS and is now recognized as existing as an entity itself, not necessarily progressing to the full-blown disease. The presence of 2 or more of these for at least 3 months, associated with a life-style risk factor, constitutes what has variously been referred to as early or pre-AIDS, AIDS prodrome, forme fruste of AIDS, or the ARC. It may or may not progress to AIDS. We accept this definition if the patients also have 2 or more defined immune abnormalities without another known cause for immunodeficiency. The immune tests used to define immune abnormalities include delayed hypersensitivity to recall antigens, absolute T-cell count, T-helper cell count, helper/sup pressor ratio, lymphocyte proliferative response to mitogens, and natural killer cell activity (see "Materials and Methods"). We INTRODUCTION AIDS3 has become a major health problem in the United States 1Supported by Grants CA-34674 and CA-31544 from the National Cancer Institute, and by grants from the President's Development Fund of M. D. Anderson Hospital and the Kaposi's Sarcoma Foundation of Houston. 2 To whom requests for reprints should be addressed, at Clinical Immunology, Box 41, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030. 3 The abbreviations used are: AIDS, acquired immune deficiency syndrome; ARC, acquired immune deficiency syndrome-related system complex; BUN, bloodurea-nitrogen; DTH, delayed-type hypersensitivity; KS, Kaposi's sarcoma; Ol, op portunistic infection; PHA, phytohemagglutinin; PWM, pokeweed symptom-free. Received May 31, 1984; accepted September 4, 1984. CANCER mitogen; SF, RESEARCH only require that these be present once. We have hypothesized that the following sequence of events may occur in the development of this syndrome. First, there is the presence of a life-style or group of risk factors which predis pose to some degree of immune deficiency. Then the patient becomes infected with the presumed transmissible causative agent. Following this, there are manifestations of a prodromal phase and, finally, the fully expressed disease entity with Ol, KS, or both. We felt that immunological characterization of patients at the beginning of this sequence might be useful. It might provide clues to early detection, mechanism of disease pathogenesis, VOL. 44 DECEMBER 1984 5894 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. IMMUNOLOGICAL CHARACTERIZATION and identifiable factors to be used as diagnostic, prognostic, and therapeutic targets. Such a study was made possible by the establishment of an AIDS clinic in the Department of Cancer Prevention at M. D. Anderson Hospital. Preliminary evaluation of the first 33 cases (30) showed that a defined complex of immune abnormalities was detectable in the "early" patients as well as those with KS and/or Ol. Complete immune evaluation has now been carried out in the first 135 of these cases and is reported herein. The study confirms a defined complex of immune abnor malities which is present in the asymptomatic high-risk patients and is most severe in those with KS or Ol. Relationships among certain factors were found and will be described. The data provide a useful set of criteria for diagnostic, prognostic, and therapeutic studies and prove that defined immune abnormalities precede the development of AIDS. MATERIALS AND METHODS All patients were referred to one of us by their physician from January 3,1982, to October 1,1983, because either AIDS or ARC was suspected or because the patient, knowing the relationship between AIDS and the homosexual life-style, asked for referral. Evaluation included a complete history, physical examination, a detailed life-style questionnaire, and standard medical as well as extensive immunological tests. All proce dures were approved by both the Institutional Research and Protection of Human Subjects (Surveillance) Committees. All subjects gave their informed consent for the evaluation, and all clinical and laboratory data were maintained in confidence. Of the 135 male subjects, all were sexually active homosexuals, 28 were asymptomatic (also referred to in this paper as SF), 74 had 2 or more of the AIDS-related symptoms (fever, night sweats, lymphadenopathy, diarrhea, weight loss, and fatigue) without cancer or Ol (referred to as ARC), and 33 had either KS, Ol, or both. These subjects averaged 34 years of age and averaged 2.4 sexual partners/week; 51.6% practiced anal intercourse as active participants and 67.7% as passive participants; 19.4% practiced active fisting and 12.8% passive fisting; 58.1% used marijuana, 71.0% nitrites, and 15.0% other recreational drugs. Prior syphillis was noted in 67%, prior gonorrhea in 79%, and prior hepatitis in 54%. The distribution of life-style factors and associated diseases among the 3 groups and their relationship to AIDS will be reported separately. Conventional medical tests included complete blood count; differential and platelet count; absolute granulocytes, lymphocytes, and monocytes; and SMA 1260 which includes total protein, albumin, globulin, calcium, phosphorus, BUN, creatinine, bilirubin, alkaline phosphatase, lactic dehydrogenase, and serum glutamic-oxaloacetic acid transaminase. Other tests included serum protein electrophoresis, quantitative immunoglobulins, and measurement of serum antibody titers to Epstein-Barr virus cytomegalovirus, and hepatitis viruses. Immunological tests carried out are listed below. All have been de scribed previously by our laboratory in the literature (30). They include: DTH skin testing by intradermal injection of the recall antigens derrnatophytin, Candida, streptokinase-streptodomase(Varidase), mumps, and purified protein derivation (30); and DTH skin testing with the new Multitest Skin Test Device (Merieux Institute) which delivers antigens via 8 tine multipuncture heads (22). This device administers trichophytin, Candida, tetanus, diphtheria, Streptococcus, Proteus, and tuberculin antigens as well as a glycerol control with a single stroke. Peripheral blood lymphocytes were evaluated for the percentage and absolute number of E-rosette-forming cells (cell-forming rosettes with neuraminidase-treated sheep RBC), cells with the surface antigens T11, T3, T4, and T8, and cells with the surface ¡mmunoglobulins IgM and IgD (17). These were determined using the Ortho-mune monoclonal antibodies and with Fab'2 polyclonal antibody to IgM and IgD from Cappel Labora tories. Since OKT11 recognizes the E-rosette receptor and since we have found that the percentage of cells positive for cell-forming rosettes with neuraminidase-treated sheep RBC and OKT11 were identical, they are used interchangeably. The percentage and number of esterase plus monocytes were determined by a nonspecific esterase stain. Peripheral blood lymphocyte proliferative responses to PHA, concanavalin A, and PWM were evaluated by tritiated thymidine incorporation after 3 days in culture (25), natural killer cell cytotoxicity (33), and antibody-dependent cell-mediated cytotoxicity (18) to human RBC and the CEM T-leukemia cell line were determined. Monocyte adherence (the numbers of mono cytes which adhere to plastic and transform to macrophages in 7 days) was also determined (15). Serum lysozyme was measured using the Worthington Biochemicals kit. Dr. Allen Goldstein (George Washington University) measured the serum thymosin «i by a radioimmunoassay (26, 29), and Dr. Olivia Prebble (Uniformed Services Medical Center) measured the serum level of a-interferon (6). Control subjects consisted of heterosexual male and female hospital employees, faculty, and medical or graduate students in the same age range as the patients. All controls were evaluated concurrent with the patient groups during the 9 months during which the study was con ducted. There were 192 subjects evaluated for WBC and differential and for leukocyte surface markers. There were 58 subjects evaluated for lymphocyte blastogenic responses. Forty subjects were evaluated for cell-mediated cytotoxicity, 37 for monocyte adherence, 47 for serum lysozyme, and 50 for thymosin «,.For serum a-interferon, controls were not run, since this activity is found in less than 3% of the sera of healthy normals. Delayed-type hypersensitivity to recall antigens was evaluated in 40 normal subjects. Two well-established statistical packages were used via the Cyber 600 main-frame computer of the University of Texas Health Science Center at Houston, SPSS and MINITAB. Distribution of patients and normals according to arbitrary points of discrimination for each laboratory value were evaluated by the x2 test. Through this, the 3 patient groups were compared to each other, the 3 groups were compared individually to the controls, and all 135 patients were compared to controls. In addition, the frequency distributions of the groups were compared by the Anova and Kruskal-Wallis one-way analysis of variance for nonparametric values. The relationship of patient clinical and immunological values to each other were tested by the Spearman Rank correlation test. For the latter, because of the large number of variables under study, only r values with p values sO.01 and r values with p values between 0.01 and 0.03 were considered significant and are reported in this paper. RESULTS A comparison of the 31 relevant hematological and immuno logical values of all 135 patients with those of the normals is given in Table 1. Chemical (SMA 1260) values are not shown, since they were essentially normal in the patients. Also given are the percentage of patients whose values were completely out side of the normal range for our laboratories or greater than 2 S.D.s from the normal mean. The 135 patients, as a group, showed significantly increased percentages of and absolute val ues for granulocytes and significantly reduced percentages of and absolute values for lymphocytes compared to the normal controls. While not significantly reduced, the hemoglobin and hematocrit values were below the normal range in about 20% of the patients, while the platelets were below the normal range in only 8% of the cases. These data indicate a potential important hematological abnormality in patients with AIDS, ARC, and the symptom-free status. OKT11, OKT3, OKT4, and OKT8 from Ortho Diagnostics, Raritan, NJ, CANCER RESEARCH OF AIDS Evaluation of the cell surface markers on peripheral blood lymphocytes revealed that the percentage of E-rosette-forming VOL. 44 DECEMBER 1984 5895 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. IMMUNOLOGICAL CHARACTERIZATION OF AIDS Table 1 Mean host defense and related parameters in patients and normals Serum levels of thymosin a, were elevated in virtually every patient in whom it was measured and was highly significantly of abnor abnormal compared to the controls. The monocyte adherence malpatients16.045.020.550.022.630.811.124.016.08.033.633.040.842.068.552.061.610.063.837.85.016.03.05 of the patients was depressed compared to normal (p < 0.01). Parameters for which at least 50% of the patients were abnormal included percentage of lymphocytes, percentage of and absolute T4+ cells, percentage of T8 cells, T4/T8 ratio, monocyte adherence, and serum thymosin a, level. Table 2A outlines the skin test reactivity to the conventional skin test battery of the total patient group compared to normal. Both the response score which converts the skin reaction sizes to an arbitrary scale and sums them, and the number of positive skin tests were significantly lower in the patients compared to the normals. Among the individual skin tests evaluated, only the mumps reaction was significantly reduced in the patients com pared to the controls. Forty % of the patients were completely anergic, while an additional 16% had only one positive test. Table 2B shows similar data on DTH using the multitest skin test battery. This battery was more sensitive than the conven tional battery in detecting the immunodeficiency associated with ParameterWBC(cummx 10")% PMNAbsolute of (percu PMN 103)% mm x lymphocyteAbsolute of lymphocytes% monocylesAbsolute of monocytesHgb(g/dl)Hct (%)Platelets mmX103)% (per cu E-rosettes(T11+)Absolute of (percu T11-1O3)%mm x 1 T3+Absolute of T3+% T4+Absolute of T4+% T8+Absolute of T8+T4/T8% M+D+Absolute of M+D+ADCC-HRBC Table 2 Delayedhypersensitivity responses Skin test diameters are mean values for mm induration. Values shown are 48hr readings. ofTC (% lysis)ADCC-CEM ofTC (% lysis)NK-K562 Parameter TClysis)Cont (% of Patients Normals A. Responsesto the conventionalskin test battery Response score 8.4 11.7 No. of positive/5 1.3 2.2 Dermatophytin(diameter) 3.4 3.3 Candida (diameter) 11.8 11.5 Streptococcus (diameter) 9.4 12.8 Mumps (diameter) 9.1 15.4 PPD (diameter) 2.8 2.0 blastogenesis(cpmx 103)PHA blastogenesisPWM blastogenesiscon-A blastogenesisMonocyte adherenceSerum p value <0.01 <0.01 NS" NS NS 0.05 NS B. Responsesto the multitest battery No. of positive/7 1.5 4.1 <0.01 Tricomonas(diameter) 0.6 1.7 <0.01 a,dig/ml)Patients6.163.94.328.82.15.30.3514.643.7263.268.11.462.11.324.40.5736.10.720.8214.00.2710.151.914.91.080.034.737.69.111.62430Normals6.559.13.936.32.34.30.2814.0-18.040.0-54. thymosin Candida (diameter) 2.3 4.1 <0.01 Streptococcus (diameter) 1.1 2.9 <0.01 " NS, not significant; PMN, polymorphonuclear neutrophilic leukocytes; Hgb, Diphtheria(diameter) 1.2 2.9 <0.01 hemoglobin; Hct, hematocrit; ADCC. antibody-dependent cellular cytotoxicity; Tetanus (diameter) 2.6 5.3 <0.01 HRBC, human red blood cells; Cont, controls; con A, concanavalin A; NK, natural Proteus (diameter) 1.1 2.3 <0.01 killer. Tuberculin (diameter) 0.9 2.4 <0.01 * NS, not significant; PPD, purified protein derivation. lysozyme(eg/ml)Serum cells was normal, while the absolute level was significantly re duced. Levels (both relative and absolute) of OKT3- and OKT4positive cells were significantly reduced, while the percentage of OKT8-positive cells was significantly increased. In contrast, the absolute level of OKT8+ cells was in the normal range. The ratio of helper (T4+) to suppressor (T8+) cells was inverted, being 0.85 in the patients and 1.85 in the controls. The percentages and absolute values for B-cells (surface ¡mmunoglobulin M+0 positive) were normal in the patient. Evaluation of cell-mediated cytotoxicity (natural killer and an tibody-dependent cellular cytotoxicity) showed no differences between patients and controls. The lymphocyte blastogenic re sponses of the patients were markedly depressed compared to the normals (p < 0.01 for each of the 3 mitogens). The thymidine incorporation of the unstimulated lymphocytes was the same in the patients and normals. Serum lysozyme was significantly elevated in the patient (p < 0.01). No patient had impaired renal function or an elevated BUN or creatinine which can explain elevated lysozyme, since this enzyme is excreted in the urine. CANCER RESEARCH Tabled Serum thymosin a, levels All patient groups were significantly higher than control; p < 0.01. Thymosin a, (pg/ml) GroupSymptom-free S.D.2387 ± ARC AIDS ControlMean ±698 990-4050 2479 ±725 2516 1191-4590 2413 ±655 2357 272-702 456 ±138Median2260 416Range1130-3687 Table 4 Circulating interferon in symptom-free, ARC, and AIDS patients Data shown are x2 between all groups using a 2 x 4 table; p < 0.001. Classification of disease Serum interferonPresent + ARC0 AbsentTotalSF VOL. 44 DECEMBER 6666KS3 O + I7 69Ol9 110KS 18Total19 1984 5896 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. 7493 IMMUNOLOGICAL CHARACTERIZATION OF AIDS Tables Mean host defense parameters in 3 patient groups compared to normals Asymptomatic Symptomatic Parameter Value WBC 103)%(per cu mm x PMNAbsolute of 103)% PMN (per cu mm x lymphocytesAbsolute of lymphocytes% monocytesAbsolute of monocytesHgb(g/dl)HctPlatelets%ofE-rosettes(T11+)Absolute KS/OI Value Value Normal value 03)% T11+ (per cu mm x 1 T3+Absolute of T3+%ofT4+Absolute T4+% T8Absolute of T8+T4/T8% M+D+Absolute of M+D+ADCC-HRBC lysis)ADCC-CEM (% of TC lysis)NK-K562(% of TC lysis)Cont (% of TC 103)PHA LBR (com x LBRPWM LBRcon-A LBR7.067.64.926.11.74.20.3415.043.028666.81.559.91.328.70.6429.60.621.1114.10.2411.750.714.51.076.440.936.7NS"<0.010.02<0.01NSNSNSNSNSNSNSNS0.02NS<0.01<0.01NSNS<0.01N Monocyte adherence 12.6 9.1 NS 9.1 NS 13.2 Lysozyme (jig/ml) <0.01 2387 2479 Thymosin a, (ng/ml) 8 All p values compare patient groups to normal controls. 0 NS, not significant; LBR, low Wastogenicresponse. For other abbreviations, see Table 1. AIDS. In addition to a significant difference in the number of positive tests per 7, the individual tests were each significantly lower in the patients compared to the controls. Table 3 outlines the serum thymosin <*i values. There was evaluation of thymosin «,in almost all of the patients. This value was elevated regardless of the clinical status and was as ele vated in the asymptomatic subjects as in those with ARC and AIDS. There was almost no overlap with the controls. Control values on Houston normals and on Washington, DC normals were comparable.4 Table 4 gives the relevant data on the serum a-interferon in the patients. Of the 93 patient sera evaluated, only 19 were found to be positive. All of these positive sera were from patients with either KS, Ol, or both. None of the asymptomatic or ARC patients had detectable serum interferon levels. It is of interest that virtually all of the Ol patients with or without KS were positive (15 of 18), while only 3 of 9 KS patients who did not have Ol showed this finding. Table 5 compares the 3 patient groups (SF, ARC, and AIDS) individually to the normals. The mean values for each group as well as the degrees of significance, determined by the KruskalWallis one-way analysis of variance, are given. Eleven of 31 parameters were significantly different than the controls among the asymptomatic subjects, 19 among the symptomatic subjects, and 19 among those with KS and/or Ol. If one also includes the <0.01 4.8 <0.01 15.9 <0.01 <0.01 10.5 2412 <0.01 <0.01 7.36 456 presence of circulating interferon, then 20 parameters were abnormal in the latter group. The relative and absolute levels of granulocytes were increased in the SF patients, suggesting both infection and the capacity of the marrow to respond. Relevant values which were not reduced in the asymptomatic group included the absolute lymphocyte count, relative and absolute levels of E-rosette-forming cells, serum lysozyme, and monocyte adherence. These values were significantly abnormal compared to the controls in one or both of the 2 other groups. These patients showed depressed or abnormal percentage of T3, per centage of and absolute T4-positive cells, a significantly de pressed T4/T8 ratio, low blastogenic responses, and an elevated thymosin at level. Blood chemistries were normal in all 3 patient groups. In general, immunological abnormalities became more severe as one moved from SF to ARC to AIDS for each of the parameters for which abnormalities were found. Table 6 compares the skin test reactivity of the 3 groups of patients with each other and the controls. In general, skin test reactivity declined in the AIDS compared to ARC and ARC compared to SF patients. The AIDS patients had very little skin test reactivity. The reactions to the multitest battery were much more sensitive than to the conventional battery in detection of immune deficiency in these patients. The 3 groups differed from each other significantly for each skin test parameter by x2 test Table 7 shows the evaluation of the differences between the 3 major patient groups. The groups were compared for each 4A. Goldstein, personal communication. CANCER RESEARCH VOL. 44 DECEMBER 1984 5897 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. IMMUNOLOGICAL CHARACTERIZATION OF AIDS Tables Skin test reactivity at 48 hr Skin test diameters are mean values for mm induration. Values shown are 48-hr readings. AsymptomaticParameterDiameterPSymptomaticDiameterPKS/OIDiameterPNormal diameterA. batteryResponse Reactivity at 48 hr to conventional scoreNo. tests/5DermatophytinCandidaStreptococcusPPDMumps8.71.83.018.19.51.39.7<0.01NS"NSNSNSNSNS7.61.41.39.89.21.48.1<0.010.02NSNSNSNSNS5.80.61.67.36.33. of positive batteryNo. Reactivity at 48 hr to multitest tests/7TricomonasCandidaStreptococcusDiphtheriaTetanusProteusTuberculin1.80.41.91.40.92.10.91.8<0.01<0.01<0.010.05<0.01<0.01<0.01NS1.80.41.90.81.12.00.90.9 of positive "NS, not significant; PPD, purified protein derivative. Table? groupsParameterWBC% Comparison of host defense parameters between 3 patient lations of parameters which would be expected because of their normal relatedness such as WBC count, and percentage and absolute granulocyte count. It is of interest that the WBC count, its components, and the T-cell parameters tended to move together. For example, WBC count, T4 level, and T8 levels PMNAbsolute of correlated directly with each other. This meant that the inverted PMN% helper/suppressor ratio was related to a fall in both cell types lymphocytesAbsolute of lymphocytes% but a greater fall in helper cells. Another observation of interest monocytesAbsolute of was that the serum lysozyme level correlated inversely with monocytesHgbHctPlateletsE-rosettesAbsolute several of the hematological and immunological parameters. Finally, because anecdotal observations suggested lack of expression of certain markers on the lymphocytes of AIDS patients, we evaluated the percentage of T11 cells failing to E-rosettes% mark with T3 and the percentage of T11 or T3+ cells failing to T3+Absolute of mark for T4 and T8. This was done by calculating the means, T3+% T4+Absolute of medians, etc., of T11 minus T3, T11 minus (T4+T8), and T3 T4+% minus (T4+T8) for each of the groups. The medians of these T8+Absolute of increased in the AIDS cases (Table 10). This was also true when T8+T4/T8% the data were evaluated by calculating the number of cases M+D+Absolute of showing either T11 or T3 minus (T4+T8) exceeding 10%. M+D+ADCC-HRBCADCC-CEMANOVA0.03NSNSNS0.05NSNS<0.01<0.01NSNSNSNS0.050.020.04<0.01NS<0.01NSNSNSNSPx2testNS"NS0.040.020.01NSNS<0.01< DISCUSSION '' NS, not significant. For other abbreviations, see Table 1. parameter by the analysis of variance and the x2 test. The general trend was for the degree of abnormality to be worse in more advanced patients. This was significant for 20 of the 38 evaluated values. It was of interest that this was not seen with the relative or absolute level of E-rosette-forming cells and with the blastogenic responses to PHA and PWM. This was also the case for the thymosin «!levels. Table 8 gives the significant correlations at the p < 0.01 level, and Table 9 gives the same at the p > 0.01 to 0.03 level between the various study parameters in the patients. It is of interest that several of the hematological and liver function parameters cor related with the most relevant immunological values. The immune parameters were lowest where the hematological and liver func tion values were the lowest. Not shown are the obvious corre CANCER RESEARCH These studies confirm and extend the observations by us (30) and others (24, 31) on the immune and related functions of patients with AIDS and the clinical states considered to be associated with the subsequent development of AIDS. Asymptomatic homosexuals, referred because they were con cerned that their life-style (multiple sexual partners, multiple sexually transmitted diseases) might eventuate in AIDS, were found as a group to have a definable immunodeficiency consist ent with that seen in a more severe form in AIDS. Low relative levels of OKT3- and OKT4-positive lymphocytes, low absolute levels of OKT4-positive lymphocytes, and inverted T4/T8 ratio, impaired lymphocyte proliferative responses to PHA, PWM, and concanavalin A and impaired skin test reactivity to the multitest antigen battery were characteristic of this group. In addition, they showed significantly elevated serum levels of thymosin at. This confirms that a definable immunodeficiency is present in the VOL. 44 DECEMBER 1984 5898 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. IMMUNOLOGICAL CHARACTERIZATION high-risk group without evident symptoms or physical signs of Tablee Spearmanrank correlations with p < 0.01 pairHgb'-albuminHgb-%ofT4+Hgb-% Variable T8+Hgb-T4/T8WBC-absolute of ENWBC-absolute T3WBC-absolute T4WBC-absolute T8WBC-monocyte adherence% of lymphocytes-abso EN% lute of lymphocytes-abso T3% lute of lymphocytes-abso T4% lute of lymphocytes-abso T8Absolute lute disease. It will be important to follow these SF patients serially pair%ofT3-absoluteEN1>% to determine if any of those with the most severe abnormalities develop the symptomatic state (ARC) or AIDS characterized by T4%ofT3-absoluteT8% of T3-absolute KS, Ol, or both. It will also be important to follow them to see if their findings are stable or if they fluctuate with time. Finally, it T3-absoluteM+DAbsolute of will be important to relate these findings to the presence of antibody to HTLV-III and to culture evidence for those viruses T3-absoluteT4Absolute known to modulate immune response such as cytomegalovirus T3-absoluteT8Absolute and Epstein-Barr virus. In all 3 patient groups, we found that virtually all patients had T3-absoluteM+DAbsolute an elevated serum thymosin at level. The etiology of this eleva T3-T4/T8%ofT4-absoluteEN%ofT4-absoluteT3% tion is obscure. We postulated previously end-organ failure with a loss of feedback regulation as the cause (21). However, Dardenne ef a/. (5) have recently demonstrated a low facteur thymique serique level in AIDS patients by a functional assay. Other possible causes include leakage from a damaged thymus or ectopie production. The functional role of the thymus and its hormones in AIDS needs to be elucidated for 2 reasons: (a) a unique form of thymic involution has been described in AIDS (7); and (b) thymic hormones are available for therapy of the syn drome (25) if a suitable rationale can be established. At any rate, whatever is responsible for the etiology of the syndrome, it must act early on thymosin a, levels, since these are abnormal in the asymptomatic group. While our controls were normals, other investigators have found thymosin o, to be normal in hospitalized cancer patients and in patients with infections.4 T8% of T4-absolute T4-absoluteM+D%ofT4-T4fT8Absolute of lymphocytes-absolute ENAbsolute lymphocytes-absolute T3Absolute lymphocytes-absolute T4Absolute lymphocytesT8% absolute T4-absoluteT8Absolute T4-absoluteM+DAbsolute T4-T4/T8% T3%ofT8-T4fT8Lysozyme-% of T8-absolute T3% of EN-absolute T4% of EN-absolute T8Absolute of EN-absolute OF AIDS ENLysozyme-%of T3Lysozyme-%of T4Albumin-% of In contrast to the above, elevated serum interferon levels T3AbsoluteEN-absolute T4Albumin-absoluteM+DCalcium-absolute of T4AbsoluteEN-absolute found only in the patients with KS, Ol, or both. In fact, only EN-absolute T8r0.490.25-0.260.250.310.370.290.280.330.630.490.400.480.890.800.700.790.440.440.410.790.700.80Variable were in Ol were detectable levels found in the majority of patients. This suggests that it is a late event, is not related to etiology, and lym therefore is a response to the disease status, and it is an phocytesCalcium-absolute ENCalcium-absolute associated phenomenon rather than a causative factor. How T3Calcium-absolute ever, its elevation has been proposed as a diagnostic test in the T4Cateium-T4/T8Cateium-con-ACalcJum-monocyteadherenceLDH-T4/T8SGOT-ADCC-CEMSGOT-thymosin disease (8). Host defense parameter abnormalities were found in signifi cantly greater frequency and at a significantly greater degree of severity in the "prodromal" patients and to an even greater degree in the patients with KS and/or Ol compared to the symptom-free patients. This suggests either that the more ¡m- a,r0.350.490.470.490.820.810.600.270.380.570.270.440.770.590.560.670.28-0.54-0.37-0.28-0.260.250.450.310.290.280.320.270.310.41-0. munologically impaired patients are the ones that progress to * For abbreviations, see Table 1. 6 EN, E-rosette-forming cells; SCOT, serum glutamic-oxaloacetic acid transam- inase. Table 9 Spearman rank correlations with p values > 0.01 and < 0.03 Variable pair r % of lymprtocytes-monocyte adherence % of ErAabsolute lymphocytes PHA B1-T4/T8 Lysozyme-absolute EN Lysozyme-absolute T4 Lysozyme-T4/T8 Albumin-absolute T3 Albumin-absolute T4 Albumin-T4/T8 Albumin-con-A Calcium-% of T8 Calcium-PWM B1 LDH-absotute T4 8 EN, E-rosette-forming cells. For other abbreviations, see Table CANCER 0.29 0.27 0.27 -0.27 -0.26 -0.20 0.25 0.24 0.20 0.26 -0.20 0.28 -0.22 1. RESEARCH AIDS or that, when AIDS occurs, it further suppresses the host defense mechanisms. Serial follow-up studies should elucidate this point. A combined effect is hypothesized, since many of the AIDS-associated infections are known to suppress cellular im munity. Other investigators have reported an absolute increase in T8+ suppressor cells in ARC patients (9). This was not found in any of our patient groups. The reason for this is unclear at present. Another interesting aspect of our subset data is that T4+T8 did not add up to T11 or T3 in the AIDS patients, suggesting an increase in "null" cells. This would be consistent with diminished mature and increased immature lymphocytes in the blood of these patients. The concurrent low monocyte adherence and high serum lysozyme deserve special comment. We speculate that the peripheral blood is depleted by the need for macro phages in infected tissues which results in redistribution of these cells. Since they become activated by AIDS and/or its associated infection, the serum lysozyme becomes elevated. The highest values were observed in the middle patient group (ARC), sug- VOL. 44 DECEMBER 1984 5899 Downloaded from cancerres.aacrjournals.org on May 19, 2016. © 1984 American Association for Cancer Research. IMMUNOLOGICAL CHARACTERIZATION OF AIDS TabtelO Defects in cell surface marker expression in AIDS and related disorders x* is used for T4+T8 - EN" normal versus ARC + AIDS,p = 0.03 and T4+T8 - T3 normal versus ARC + AIDS,p = 0.04. Parameter(% of positive cells) Groups EN-T3 (median) EN-T4+T8 (median) T3 - T4+T8 (median) No. of cases showing T4+T8 -EN*10%(%) NormalSymptom-freeARCAIDS-3.33.01.83.5-1.14.04.612.01.13.32.61.56 (5)1 (4.5)9(13.2)4 No. of cases showing T4+T8 -T3s10%(%) (4.2)2 (9.0)6 (8.8)5(16.1) (12.9)5 "EN, E-rosette-forming cells. gesting that this group can be maximally activated after which there may be a failure of the bone marrow to provide enough cells for further activation. The high serum lysozyme was not due to a renal failure, since all BUN and creatinine values were normal in these patients. The correlations between hematological and chemical values on the one hand and immunological values on the other are also worthy of special note. In general, the hemoglobin and WBC count values and the albumin and calcium values correlated directly and significantly with such immune parameters as the level of T4+ cells and the helper/suppressor ratio. This means that high values of one correlated with high values of the other. The correlation of the T4+ cells and particularly the T4/T8 ratio with functional immune parameters such as lymphocyte prolif eration and DTH suggests a possible causitive relationship. We have demonstrated previously that these patients have suppres sor cell activity and it correlates with their helper/suppressor ratio (16). The correlation between the hematological and im mune parameters may be on the same basis, namely, suppressor cell activity in the marrow. Immune regulation in normal marrow does occur (23) and has been suggested as playing a role in this disease (24). The concurrent low monocyte adherence and high serum lysozyme deserve special comment. We speculate that the pe ripheral blood is depleted by the need for macrophages in infected tissues. We suggest that this and the significant decline in blood esterase-positive cells are due to a predominance of tissue localization and activation of macrophages, a change in monocyte kinetics and, late in the disease (in patients with KS or Ol), a diminished marrow production of monocytes. Elevated serum lysozyme is seen in both animals (4) and humans (19) after administration of macrophage activators, and abnormally low levels are noted in disorders of monocyte deficiency such as hairy cell leukemia (20). It may also be that the activation of monocytes results in monocyte-mediated suppression of im mune responses. In this study, we have evaluated hematological, chemical, and immunological parameters in patients with AIDS, in its "prod rome," and in symptom-free subjects with a high-risk homosex ual life-style. A number of parameters were significantly abnormal in asymptomatic subjects who belong to the high-risk group and worsen significantly in the other 2 groups. Several parameters (serum thymosin a1f PHA, and PWM responses, certain skin tests) are equally abnormal in all 3 groups. About 20 parameters are abnormal in the ARC and AIDS patients. One parameter (serum interferon) is abnormal only in the KS and Ol patients. These results have defined the spectrum of disease quantita tively and have provided a data base for pathogenetic and CANCER RESEARCH prognostic studies and for targeting and monitoring therapeutic approaches. REFERENCES 1. Centers for Disease Control. 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