Multitest CMI for standardized measurement of delayed cutaneous hypersensitivity and cell-mediated immunity. Normal values and proposed scoring system for healthy adults in the U.S.A

The Multitest CMI system, a disposable device that simultaneously applies seven standardized preloaded antigens and diluent control, is a major advance for measurement of delayed type hypersensitivity (DTH) in assessment of cell-mediated immunity (CMI). The system was tested in 402 healthy adults, aged 17 to 92 years, to determine normal values for incidence and size of DTH responses. Incidence of positive responses to individual antigens varied from 85% to 46%, with great variability related to age and sex. To better assess CMI, a two-part score based on 48-hour readings was employed. The mean number of positive antigens ranged between four and five, and the mean sum of their mm induration ranged between 18 and 25, with both scores increasing with advancing age. A statistical zone of reduced DTH scores (hypoergy) was identified. The Multitest CMI system appears to be a practical means of reproducibly assessing CMI in subjects with immunologic, metabolic, infectious, or neoplastic disorders. The scores in our population may serve as reference values to which results from any tested adult can be compared.     

Assessment of cell-mediated immunity in a British population using multiple skin test antigens

An in vivo method of assessing the competence of the cell-mediated immune system (Multitest CMI) was used in 200 healthy volunteers (age range 17–88 years). The profile of reactivity to seven individual antigens was determined. A positive reaction was obtained in 96·5% of the subjects who reacted positively to at least one antigen with 78% reacting to two or more antigens. The number of positive responses and the degree of reactivity was significantly reduced in elderly subjects and in females aged 17–65 years. The Multitest CMI system provides a rapid and convenient method of assessing cell-mediated immunity (CMI) in vivo and could have a wide range of applications in the investigation of immunological, infective and neoplastic conditions.     

Delayed hypersensitivity responses of cancer patients to recall antigens using a new "Multitest" applicator

Testing of delayed-type hypersensitivity to recall antigens is acknowledged to be a useful assessment of cell-mediated immunity in cancer patients. The conventional method, however, has suffered from a lack of standardized antigens and variability owing to the method of application of the recall antigens. The Multitest-CMI system by the Merieux Institute offers an alterative. It employs standardized antigens and a single-stroke applicator, which is simpler to use and presumably subject to less variation. The present study compares the conventional and Multitest batteries done over an 8-month period in 403 cancer patients. The results suggest that the Multitest is easier to administer and is tolerated better by patients than the conventional method. Furthermore, the Multitest can detect recall antigen anergy or reactivity and correlates well in degrees of reactivity to the individual test battery. Consequently, Multitest-CMI can be substituted for the conventional battery in skin testing for immunocompetence in cancer patients.     

Cell-mediated immunity assessed by skin testing (Multitest®)

In 60 healthy adult volunteers and 58 patients with gastrointestinal disease a test system (Multitest) consisting of a plastic disposable multiple-puncture device capable of simultaneously applying seven delayed-type hypersensitivity antigens and a glycerin/saline diluent (negative control) was assessed. The Multitest device was applied on both the inner side of the forearm and on the back for assessment of cell-mediated immunity (CMI). The antigens used were two toxoids, tetanus and diphtheria, three bacterial antigens, Streptococcus, tuberculin and Proteus and two fungal antigens, Candida and Trichophyton. A scoring system based on both number and size of positive response revealed a median "score" on arm and back of 19 mm and 14 mm respectively, in the healthy volunteers and a median "score" of 12 mm and 8 mm respectively in patients with gastrointestinal disease. In both groups a significant difference was found between back and arm (P less than 0.01). The coefficient of determination (r2) shows that only 64% of the variability in scores on the back is explained by the regression line. Therefore, scores obtained from tests on the back cannot be interpreted with reference to normal values originating from tests applied to the inner side of the forearm.     

Serial delayed cutaneous hypersensitivity skin testing with multiple recall antigens in healthy volunteers: booster effect study

Booster effects on delayed cutaneous hypersensitivity (DCH) responses have been demonstrated for various antigens when DCH is measured by the Mantoux technique. In the present study, we investigated this possibility when assessing DCH responses using the Multitest CMI multipuncture technique with simultaneous injections of seven test antigens and a control. The DCH responses were quantified for each antigen and for the overall DCH response expressed as a DCH score. In a group of healthy volunteers, DCH was repeatedly tested either 1 month apart or 2 months apart at least six times. When volunteers remained healthy, DCH variations were observed with only two of seven tested antigens: streptococcus which slowly decreased (P = .012) and proteus which slowly increased (P = .04). Responses to the other antigens and the DCH score remained stable. In contrast, greater DCH variations were observed when infections occurred. The results with the Multitest CMI multipuncture show that repeated application had minimal booster effect on DCH responses and may be used to evaluate and follow immunocompetence of patients.     

Three suitable antigens for delayed-type hypersensitivity skin testing in a tropical country like Thailand

Delayed type hypersensitivity (DTH) skin test is a standard tool to assess in vivo cell-mediated immunity. Mantoux method using 4-5 common recalled antigens is recommended. However, not all antigens are widely available and appropriate antigens for tropical countries are not known. The objective of this study is to investigate what and how many antigens should be included in the DTH testing panel that suitable for Thailand and may be for this region. The DTH skin tests were done by Mantoux method in a double blinded fashion. Average induration size of > or = 5 mm defined as a positive test. Antigens included purified protein derivative (PPD), Candida albicans, tetanus toxoid (TT), Trichophyton mentagrophytes and hepatitis B vaccine (HBV). The negative control was normal saline. Of 95 healthy subjects, all showed DTH positive to > or = 1 antigen. The positivity to C. albicans, tetanus toxoid, PPD, T. mentagrophytes, and HBV was 92.6%, 83.2%, 82.1%, 50.5%, and 5.3%, respectively. When three antigens: PPD, TT and C. albicans were analyzed, 100% of subjects showed a positive response to > or = 1 antigen and 96.8% showed a positive response to > or = 2 antigens. When only PPD and TT were analyzed, 100% of subjects showed > or = 1 antigen positive and 68.4% showed both antigens positive. C. albicans antigen at 1:100 was associated with a high incidence of fever (2/20) and large local reaction (7/20), 1:500 was found to be the optimal concentration. PPD, TT and C. albicans are suitable to be included in a DTH skin testing in a tropical country like Thailand. However, in a setting where C. albicans extract is not available, testing with only two antigens of PPD and tetanus toxoid may be an alternative, but with a lower sensitivity.     

Gender differences in response to the multitest CMI skin test in the general population.

BACKGROUND: Previous studies of gender differences in response to the Multitest CMI skin test have produced conflicting results. OBJECTIVE: To determine whether gender is associated with response to the Multitest CMI skin test. METHODS: Two-hundred ninety-seven adults, aged 18 to 64 years, recruited originally for a study of the immune effects associated with living near a hazardous waste site containing primarily organochlorine pesticides, underwent a skin test using the Multitest CMI skin test. Six of seven antigens were tested: tetanus toxoid, diphtheria toxoid, Candida, Tricophyton, Streptococcus, and Proteus. The tuberculin antigen was excluded. Lymphocyte function was also evaluated in vitro using standardized methods of mitogen stimulation with phytohemaglutinin (PHA), concanavalin A (CON-A), and pokeweed mitogen. RESULTS: The frequency of positive responses to the skin tests was significantly (P < .001) higher among males (80.4%) than among females (55.7%). Males were more likely than females to respond to all six antigens tested (P < .05). The mean diameter of positive skin test measurements for males statistically significantly (P < .05) exceeded female responses for tetanus and diphtheria. Although not statistically significant, male response size exceeded that of females for all other antigens except Trycophyton. Controlling for age, race, smoking, income, and plasma DDE levels did not change these results. Skin test positivity was not associated with mitogen stimulation assay results overall or within gender groups. CONCLUSION: Significant gender differences in response to the Multitest CMI skin test could limit its use as a marker of anergy in general population studies.     

Declining cell-mediated immunity and increased chronic disease burden.

BACKGROUND: The effects of chronic disease have been proposed as an explanation for conflicting results in studies of age effects on cell-mediated immunity. OBJECTIVE: To examine the hypothesis that declining cell-mediated immunity is more closely linked to chronic disease burden than to chronological age. METHODS: Fifty-eight elderly individuals were tested for delayed-type hypersensitivity (DTH) responses to Candida and tetanus antigens. Disease burden was quantified using the Cumulative Illness Rating Scale (CIRS). Higher CIRS scores reflect greater disease burden. Mean DTH response by age group (<71, 71-78, and >78 years) was compared with mean DTH response by CIRS score (<11, 11-15, >15). Total serum IgE levels were measured and similarly stratified by age and CIRS score. RESULTS: Mean Candida DTH responsiveness declined progressively with increasing disease burden (increasing CIRS score). Mean DTH responses were 7.78, 3.05, and 0.0 mm for CIRS scores less than 11, 11 to 15, and greater than 15, respectively. Candida DTH responses showed no progressive decline with advancing age. Mean DTH responses were 4.7, 3.5, and 5.0 mm in participants younger than 71, 71 to 78, and older than 78 years, respectively. Total serum IgE levels increased with advancing age. Mean total IgE levels were 182, 249, and 342 IU/mL in participants younger than 71, 71 to 78, and older than 78 years, respectively. No correlation was observed between mean total IgE levels and CIRS scores. CONCLUSIONS: An inverse relationship between Candida DTH response and CIRS score suggests that increased chronic disease burden is associated with diminished cell-mediated immune response. Advancing age did not predict a diminished DTH response in our patients. No relationship was observed between chronic disease burden and total serum IgE level.     

Parameters of T cell mediated immunity to commensal micro-organisms in patients with chronic purulent rhinosinusitis: a comparison between delayed type hypersensitivity skin test, lymphocyte transformation test and macrophage migration inhibition factor assay.

In 75 patients with unexplained chronic purulent rhinosinusitis T cell mediated immunity to three micro-organisms frequently colonizing the human upper respiratory tract, viz. Haemophilus influenzae, streptococci and Candida albicans, was assessed. Delayed type hypersensitivity (DTH) skin test reactivity was measured in vivo, whereas the blastogenic responsiveness (lymphocyte transformation test; LTT) and lymphokine production (e.g. migration inhibition factor; MIF) of the lymphocytes upon antigen stimulation were measured in vitro. MIF was assayed with a recently developed test system using the human monocytoid cell-line U937 as indicator cells in agarose microdroplets. Two-thirds of the 75 patients tested showed a defective DTH response to one or more of the microbial antigens; this contrasted to the findings in 25 healthy subjects, of whom over 90% showed a positive DTH reaction to any of the three antigens. PHA skin tests were entirely normal in both patients and healthy controls. Microbial antigen-specific LTT responses fluctuated considerably in time from strongly positive to negative and vice versa in healthy individuals as well as in patients. In general however, blastogenic responses in patients were comparable to or even higher than those of healthy persons. In the MIF assay, lymphocytes of all healthy individuals tested showed production of MIF upon stimulation with all three antigens; this again contrasted to two-thirds of the patients, whose lymphocytes showed a defective MIF production. Fluctuations of MIF-production in time could not be established and a very good correlation existed between the data obtained in the MIF assay and those of the DTH skin tests. These results indicate that apart from skin testing, the MIF assay seems to be the most suitable parameter to assess defects in T cell reactivity towards microbial antigens. These defects exist in two-thirds of our patients suffering from chronic purulent rhinosinusitis.     

Cell-mediated immunity assessed by skin testing (Multitest®)

The Multitest CMI system consists of a disposable multiple puncture device that simultaneously applies seven standardized recall antigens for assessment of cell-mediated immunity (CMI). The seven antigens included were toxoid from Clostridium tetani and Corynebacterium diphtheriae, tuberculin, plus antigens from streptococcus (group C), Candida albicans, Trichophyton mentagrophytes, and Proteus mirabilis. A population of 352 healthy Danish adults, aged between 17 and 90 years, was tested to determine the incidence and size of delayed-type hypersensitivity (DTH) responses. All but six healthy adults (98%) responded to one or more antigens, the median number of positive responses being four in males and three in females. The incidence of positive responses ranged from 91% for tuberculin to 11% for trichophyton. The number of positive responses declined with age, being somewhat faster in females than males. Six of the seven antigen response rates were significantly lower in the over 65-years-olds, the only exception being trichophyton, and for four of the seven antigens significantly lower in females compared to males. When correlated to age and sex no major differences were found with regard to the size of response to the seven antigens except that the tuberculin response was larger in males. A scoring system based on both number and size of positive responses revealed significant age and sex related differences. The median "score" in 17-65-year-old males and females were, respectively, 17 mm and 14 mm compared to 13 mm and 8 mm in those over 65 years old (P less than 0.001 for both comparisons).     

Biochemical correlates of in vivo cell-mediated immune dysfunction in patients with depression: A preliminary report

We have previously demonstrated that at least 50% of patients with melancholia have impaired cell-mediated immunity (CMI) as assessed by delayed-type hypersensitivity (DTH) skin responses to a standardized battery of antigens. Hypercortisolaemia and increased circulating catecholamines both occur in patients with severe depressive disorders and each has been proposed as a possible mediator of observed immune abnormalities in patients with mood disorders. As part of a larger study, we collected 24 h urine samples from 28 patients with major depression and measured concentrations of urinary free cortisol (UFC), the noradrenaline metabolite dihydroxyphenylglycol (DHPG), adrenaline, and the dopamine metabolite DOPAC. CMI multitest skin testing revealed a reduced or absent response in 54% of subjects. Those with reduced DTH skin responses demonstrated increased urinary adrenaline (P<0.02), with trends towards increased UFC (P = 0.052) and increased DHPG (P = 0.06). These differences could not be attributed to differences in age or depression severity. Correlational analyses demonstrated inverse associations between the extent of DTH responsiveness and 24 h levels of urinary adrenaline and DHPG, with similar trends evident for UFC and DOPAC. These results suggest that both circulating catecholamines and cortisol may play roles in the reduction of CMI in patients with severe depression.     

The interaction of social network size and stressful life events predict delayed-type hypersensitivity among women with metastatic breast cancer.

This study examined relationships between social support, stressful life events and antigen-specific cell-mediated immunity. Participants were 72 women with documented metastatic breast carcinoma, who completed self-report measures of social support and life stress. Immune response was assessed using the delayed type hypersensitivity (DTH) skin test. Number of positive antigens was significantly related to the interaction of social network size and stressful life events (p<0.05). Number of positive antigens was greater for women who had experienced a high frequency of stressful life events but who reported a larger network of support. However, social network size was inversely related to DTH response among women who had experienced fewer stressful life events. Average induration size was not significantly related to the quality of social support, life stress per se, or their interactions. The relationship between social network size and immune response in women with metastatic breast cancer depends on prior stressful life experience.     

Lymphoblast proliferation and humoral antibody response after rubella vaccination

Cell-mediated immunity was assessed in man by summation of positive delayed hypersensitivity (DHS) responses to five test antigens, Candida, mumps, trichophyton, tuberculin and streptokinase-streptodornase (Varidase). The study included forty-four controls comprising healthy persons and hospital inpatients with minor illnesses, forty-five aged persons, twenty-four patients with active chronic hepatitis (ACH), nine with systemic lupus erythematosus (SLE) and fourteen patients with rheumatoid arthritis. For controls and the group with ACH, cell-mediated immunity was assessed also by transformation of blood lymphocytes with phytohaemagglutinin (PHA). The incidence of positive DHS responses to all antigens fell with advancing age, and responsiveness was significantly lower in all three groups of immunopathic disease, particularly ACH. Prednisolone was not associated with significantly depressed DHS reactions, but combined prednisolone and azathioprine were. Weak DHS responses were associated significantly with low titres of natural antibody to flagellin and with lower humoral immune responses to flagellin. Lymphocyte transformation in response to PHA in ACH was significantly less than in healthy controls. Thus certain autoimmune diseases show an immunological imbalance in the form of depressed cell-mediated immunity to extrinsic antigens and `hyperactive' humoral immune responses to `self' antigens and selected extrinsic antigens.     

Cell-mediated immunity in subjects immunized with HLA antigens

Production of cellular immune reaction in subjects immunized against HLA antigens was studied by the capillary migration inhibition test in vitro and by the skin test in vivo. The majority of the immunized subjects (10 out of 13) developed a cell-mediated immunity to the donor antigens. Production of HLA antibodies was demonstrable in a smaller proportion of the cases. The complementary nature of humoral and cellular responsiveness was borne out by the present observations. The result of the skin test performed simultaneously with the migration inhibition test correlated well with the result in vitro.     

In vivo testing confirms a blunting of the human cell-mediated immune mechanism during space flight.

The cell-mediated immune (CMI) mechanism was evaluated in 10 space shuttle astronauts by measuring their delayed-type hypersensitivity response to seven common recall antigens. The Multitest CMI test system was used to administer antigens of tetanus, diphtheria, Streptococcus, Proteus, old tuberculin, Candida, and Trichophyton to the forearm 46 h before nominal mission termination; readings were conducted 2 h after landing. The mean number of reactions was reduced from 4.5 preflight to 3.0 inflight, and the mean reaction score was reduced from 21.4 to 13.7 mm inflight. The data presented suggest that the CMI system is still being degraded by space flight conditions on day 4 and that between day 5 and day 10, the depression maximizes and the system begins to adjust to the new conditions. The relation of these in vivo findings to previously reported in vitro results is discussed.     

Cell mediated immunity in school children assessed by Multitest CMI skin testing II. Epidemiologic factors affecting immune responsiveness

The Multitest CMI system was used to measure cutaneous delayed type hypersensitivity (DTH) to a standardized battery of seven recall antigens in 448 healthy school children in relation to sex, age, race, and socioeconomic groups. Blacks demonstrated highest overall DTH, hispanics were intermediate, and whites were lowest. There was a trend for males to be more reactive than females at certain ages in each race. Incidence of reduced DTH scores (relative to adult values) was somewhat increased in the youngest school children, chiefly whites. Incidence and size of DTH responses to certain antigens were consistently higher in the blacks and/or hispanics as compared with whites. Blacks lived almost exclusively in the "poorer" school district, hispanics were evenly divided in both districts, while whites were mostly from the "affluent" district. Level of DTH appeared to be greatest in children from relatively poor homes possibly reflecting more intense and frequent exposure to ubiquitous microorganisms.     

Humoral and cell-mediated immune responses in chronic typhoid carriers.

Humoral and cell-mediated immune responses to Salmonella typhi were studied in 10 chronic typhoid carriers, and in healthy controls. Carriers showed impaired cellular reactivity to S. typhi antigens in the leucocyte migration inhibition test (LMI). Carriers did not show a generalized depression of cell-mediated immunity in that delayed hypersensitivity skin test responses to recall antigens, peripheral blood T cell numbers, and lymphocyte transformation responses to mitogens were normal. Lymphocyte transformation in the presence of S. typhi antigen occurred to a greater extent than normal in four of six subjects tested and suggested the possibility of dissociated defects of cellular immunity. Carriers showed normal humoral immunity, as judged by antibodies to the flagellar and somatic antigens of S. typhi and S. paratyphi and to Vi antigens of S. typhi. The results suggest that the carrier state may be the consequence of a specific defect in cell-mediated immune responses to S. typhi.     

A delayed-type hypersensitivity skin-test system using the insulinoma cell line RINm5F to monitor beta cell-specific cellular autoimmune reactivity in the spontaneously diabetic BB/O rat.

Islet-specific autoimmune reactivity (humoral and cell-mediated) is the basis for the insulitis process of type I diabetes mellitus. In this report a delayed-type hypersensitivity (DTH) skin test was used to monitor the presence of an islet-specific cell-mediated autoimmune component in BB/O rats. The BB/O rat is a strain characterized by the spontaneous development of type I diabetes. Intact RINm5F cells as well as a RINm5F cell membrane preparation were used as DTH skin test antigens. Rats of different ages and disease stages were tested in the ear with the insulinoma cell line and its cell membrane preparation. As control antigens, the fibroblast cell line 3Y1 and a cell membrane preparation made thereof were used. The DTH reaction system showed a positive cell-mediated reactivity in BB/O rats for membrane-bound RINm5F cell antigens, and not for the control fibroblast 3Y1 cell membrane determinants. The true DTH character of the skin test was established by the time-course of the reaction (maximum at 24 h), the histopathology (infiltration by dendritic cells, lymphocytes and macrophages), and the possibility to transfer the reaction with spleen cells and lymph node cells. The DTH test towards RINm5F cells showed the highest prevalence of positivity (100%) in BB/O rats around the onset of diabetes (3 weeks before to 3 weeks after the onset of glucosuria). The prevalence of DTH positivity was 56% in the period of more than 3 weeks before the onset of glucosuria. In BB/O rats with a duration of glucosuria of more than 3 weeks, the prevalence of positivity was around 60-70%.     

Kinetics of cellular infiltration and cytokine production during the efferent phase of a delayed-type hypersensitivity reaction.

Cell-mediated immunity is a primary host resistance mechanism against many infectious organisms and is responsible for leucocyte recruitment to the infection site. Delayed-type hypersensitivity (DTH) reactions are in vivo correlates of cell-mediated immunity and have long been used to assess the level of cell-mediated immune (CMI) responsiveness to specific antigens. It has been difficult to study the kinetics of cellular influx and cytokine composition at the site of an on-going CMI reaction. Consequently, knowledge of the sequential events occurring during the efferent phase of a CMI response is incomplete. Here we report on the use of a gelatin sponge model for evaluating the progression of events during the effector phase of a DTH reaction to antigens of the mycotic organism Cryptococcus neoformans. Previously, we have shown that 24 hr after antigen injection into sponges in infected or immune mice, the leucocyte types infiltrating the sponges are consistent with a classical murine DTH reaction. Through kinetic studies, we show here that neutrophils are the first leucocytes to appear in DTH-reactive sponges, followed by increases in lymphocyte and then monocyte numbers. Tumour necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-5 were elevated in DTH-reactive sponges compared with control sponges, and each cytokine had a relatively unique temporal profile. IL-4 was not detectable in the sponges. Together our data indicate that the expression of a CMI response comprises a well-regulated sequential influx of leucocytes that contribute to the lymphokine composition of the reaction.     

Lymphocyte responses to purified ragweed allergens in vitro: I. Proliferative responses in normal,newborn, agammaglobulinemic,and atopic subjects

To evaluate cell-mediated immune responsiveness to pollen allergens in atopic subjects, we studied the deoxyribonucleic acid (DNA) synthetic responses of their cultured lymphocytes to purified ragweed antigens E, K, and Ra-3. Since lymphocytes from some highly ragweed-sensitive subjects gave poor proliferative responses when harvested on day 6, we undertook a series of dose-response and time-course studies in atopic and control subjects. Surprisingly, vigorous DNA synthetic responses to antigen E occurred with lymphocytes from all 45 subjects, including 19 highly ragweed-sensitive atopic adults (8 immunotherapy treated, 11 untreated); 13 nonatopic controls; 4 newborns, and 9 agammaglobulinemic patients. The geometric mean of peak response counts per minute in all 45 subjects was 21,163 and in unstimulated cultures was 2,416 (p = < 0.0001). The mean day on which the maximal responses occurred was 8.7, and the mean dose eliciting the maximum responses was 59 μ/ml. Statistical comparisons of the stimulated culture data revealed no significant intergroup differences. The finding of vigorous responsiveness to these purified pollen allergens by lymphocytes from nonatopic normal, newborn, and agammaglobulinemic subjects suggests that ragweed pollen antigens are either ubiquitous and lead to cell-mediated responsiveness in all subjects with intact cell-mediated inmunity, or that they may have mitogenic properties in addition to their known antigenic properties.