New Paradigm for Translational Modeling to Predict Long-term Tuberculosis Treatment Response.

Disappointing results of recent tuberculosis chemotherapy trials suggest that knowledge gained from preclinical investigations was not utilized to maximal effect. A mouse-to-human translational pharmacokinetics (PKs) - pharmacodynamics (PDs) model built on a rich mouse database may improve clinical trial outcome predictions. The model included Mycobacterium tuberculosis growth function in mice, adaptive immune response effect on bacterial growth, relationships among moxifloxacin, rifapentine, and rifampin concentrations accelerating bacterial death, clinical PK data, species-specific protein binding, drug-drug interactions, and patient-specific pathology. Simulations of recent trials testing 4-month regimens predicted 65% (95% confidence interval [CI], 55-74) relapse-free patients vs. 80% observed in the REMox-TB trial, and 79% (95% CI, 72-87) vs. 82% observed in the Rifaquin trial. Simulation of 6-month regimens predicted 97% (95% CI, 93-99) vs. 92% and 95% observed in 2RHZE/4RH control arms, and 100% predicted and observed in the 35 mg/kg rifampin arm of PanACEA MAMS. These results suggest that the model can inform regimen optimization and predict outcomes of ongoing trials.

Comparison between a whole blood interferon-gamma release assay and tuberculin skin testing for the detection of tuberculosis infection among patients at risk for tuberculosis exposure.

A new test that measures interferon-gamma (IFN-gamma) release in whole blood following stimulation with tuberculin has the potential to detect tuberculosis infection using a single blood draw. The IFN-gamma release assay was compared with the standard tuberculin skin test (TST) among 467 intravenous drug users at risk for tuberculosis in urban Baltimore. Among 300 human immunodeficiency virus (HIV)-seronegative patients, the IFN-gamma release assay was positive in 177 (59%), whereas the TST was positive in 71 (24%), for a percent agreement of 59% (kappa=26%). Among 167 HIV-seropositive subjects, the IFN-gamma release assay identified 32 reactors (19%); the TST identified 16 reactors (9.6%), for a percent agreement of 82% (kappa=28%). The IFN-gamma release assay detected more reactors than did the TST, but its agreement with TST was weak. As the TST is an imperfect standard, further evaluation of the IFN-gamma release assay among uninfected persons and persons with culture-confirmed tuberculosis will be useful.

Pulmonary bovine-type tuberculosis in rabbits: bacillary virulence, inhaled dose effects, tuberculin sensitivity, and Mycobacterium vaccae immunotherapy.

This report elucidates four aspects of the immunology of pulmonary tuberculosis produced in rabbits: (i) the virulence of bovine-type tubercle bacilli, strain Ravenel S, (ii) systemic factors influencing the generation of visible primary pulmonary tubercles, (iii) differences in tuberculin sensitivity of rabbits and humans, and (iv) the effect of Mycobacterium vaccae immunotherapy on cavitary tuberculosis. Laboratory strain Ravenel S (ATCC 35720) was not fully virulent. Fully virulent strains produce one visible primary pulmonary tubercle for each three bacillary units inhaled. Strain ATCC 35720 produced one such tubercle for each 18 to 107 bacillary units inhaled, indicating that its virulence was reduced by 6- to 36-fold. When a low dose of this Ravenel S strain was inhaled, the host resistance (measured by the number of inhaled bacilli needed to generate one visible primary pulmonary tubercle) was increased at least 3.5-fold compared to the host resistance when a high dose was inhaled. Rabbits and humans differ in the degree and in the maintenance of their dermal sensitivities to tuberculin. Compared to rabbits, humans are 100 times more sensitive to tuberculin. Also, at 33 weeks rabbits with well-controlled cavitary tuberculosis usually showed a decrease in their tuberculin reactions of about 50% from peak values, whereas humans with such well-controlled tuberculosis are thought to maintain strong reactions for many years. These species differences may be due to desensitization to group II mycobacterial antigens in the rabbits because they have a different diet and a different type of digestive tract. M. vaccae immunotherapy of rabbits with cavitary tuberculosis produced no statistically significant effects. Experiments with many more rabbits would be required to prove whether or not such immunotherapy is beneficial.

Distress, symptom distress, and immune function in women with suspected breast cancer.

PURPOSE/OBJECTIVES: To investigate distress and its association with immune function among women with suspected breast cancer. DESIGN: Prospective, descriptive, correlational study. SETTING: An outpatient breast clinic at a tertiary urban hospital. SAMPLE: A convenience sample of women who had either a fine needle aspiration or open breast biopsy for a suspicion of breast cancer. Thirty-five women comprised the study sample, 6 with malignant and 29 with benign tumors. METHODS: Data were collected at three points in time. The first time (T1) was after the physician visit when the need for breast biopsy was ascertained. The second time (T2) was 7-10 days postbiopsy, and the third time (T3) was 7-10 days after T2. At T1, T2, and T3, participants filled out the Brief Symptom Inventory (a measure of psychological distress) and the Adapted Symptom Distress Scale (a measure of symptom distress) and provided a blood sample. Demographic data also were collected at T1. Immune function was measured by serum cytokine levels of transforming growth factor beta (TGF beta) and tumor necrosis factor alpha (TNF alpha). MAIN RESEARCH VARIABLES: Psychological distress, symptom distress, and immune function. FINDINGS: Psychological distress scores were moderate to high. Symptom distress was either nonexistent or slight. Significant correlations between psychological distress and symptom distress were found at T2 and T3. At T2, significant relationships between psychological distress and TNF alpha and between symptom occurrence and TNF alpha were found. Psychological and symptom distress scores were significantly different between women with malignant versus benign tumors at all three times. No differences in cytokine levels were found between the groups. CONCLUSIONS: These results suggest the strong effect that the diagnostic process has on psychological distress and its potential effects on immune functioning. Distress was significantly greater for women with malignant disease; however, women with benign disease continued to have elevated levels of distress. IMPLICATIONS FOR NURSING PRACTICE: Nurses should be aware of the extremely stressful nature of the diagnostic phase and should continue to provide support, knowing that this distress continues throughout this phase, particularly for women diagnosed with malignancy.

Nonspecific and immune-specific up-regulation of cytokines in rabbit dermal tuberculous (BCG) lesions.

To our knowledge, this is the first sequential study of cytokines in tissue sections of developing and healing tuberculous (BCG) lesions. In situ hybridization, immunohistochemical, and RT-PCR techniques were used. Cytokine mRNAs showed a biphasic pattern. The percentage of mononuclear cells (MN) containing IL-1beta, TNF-alpha, MCP-1, and IL-8 mRNAs was highest in 1- to 3-day lesions, apparently because of the nonspecific inflammatory response caused by the tubercle bacilli in the BCG vaccine. At 5 days, this percentage was significantly reduced. With IFN-gamma, the peak and trough were delayed by 2 days. By 9 days, the percentage of MN containing the mRNAs of all five cytokines had again increased and the rabbits had become tuberculin-positive. In general, MCP-1 and TNF-alpha proteins and the vascular adhesion molecules, ICAM, VCAM, and perhaps ELAM, peaked at about 3 days. Many mononuclear cells surrounding the central areas of solid and liquefied caseous necrosis contained chemokine IL-8 mRNA. IL-8 is known to attract PMN, and PMN were present nearby. In contrast, MN containing chemokine MCP-1 mRNA were present more peripherally in areas rich in macrophages and lymphocytes. The early nonspecific cytokine response seems to be an adjuvant effect of the mycobacteria in BCG vaccine in that it causes a rapid entry of macrophages, lymphocytes, granulocytes, and probably dendritic cells into local sites of antigen deposition. This effect should be considered in developing improved vaccines for the prevention of tuberculosis, because BCG vaccines producing a strong early cytokine response should be more immunogenic than BCG vaccines with similar antigens producing a weak response.

Comparison of a tuberculin interferon-gamma assay with the tuberculin skin test in high-risk adults: effect of human immunodeficiency virus infection.

A novel, whole blood interferon-gamma (IFN-gamma) assay was evaluated to determine its suitability for detecting Mycobacterium tuberculosis exposure in intravenous drug users with or without human immunodeficiency virus (HIV) infection. Whole heparinized blood was incubated overnight in separate wells with tuberculin purified protein derivative (PPD), saline, and mitogen controls. Levels of IFN-gamma in plasma supernatants were determined by rapid ELISA. Participants were then administered the tuberculin skin test (TST) and tested for cutaneous anergy. The whole blood IFN-gamma test agreed (89%-100%) with a positive TST in both HIV-seropositive and -seronegative subjects, but reactivity to PPD was more detectable by the whole blood assay among those with negative TSTs or anergy. TST induration diameter and IFN-gamma responses were correlated (Spearman's p = .45, P = .0001), but both responses were blunted by HIV infection. In summary, tuberculin reactivity appears to be more detectable by the whole blood IFN-gamma assay than by TST, and the assay requires no return visit for test reading.

Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli.

Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. In liquefied caseum, the tubercle bacilli grow extracellularly for the first time since the onset of the disease and can reach such large numbers that mutants with antimicrobial resistance may develop. From a cavity, the bacilli enter the bronchial tree and spread to other parts of the lung and also to other people. Of the commonly used laboratory animals, the rabbit is the only one in which cavitary tuberculosis can be readily produced. This report is the first to describe and analyze the complete course of cavitary tuberculosis, produced by aerosolized virulent bovine-type tubercle bacilli in commercially available New Zealand white rabbits. After the inhalation of 220 to 880 bacillary units, all of the rabbits were overtly well until they were sacrificed at 33 weeks. After the inhalation of 3,900 to 5,800 bacillary units, half of the rabbits died of progressive tuberculosis between 5 and 9 weeks and the other half lived until they were sacrificed at 18 weeks. Pulmonary cavities developed in both low- and high-dose groups, some beginning as early as 6 weeks. Bacilli from primary cavities sometimes caused nearby secondary cavities, but more frequently, they ascended the bronchial escalator, were swallowed, and caused secondary tubercles in the lymphoid tissue of the appendix and ileocecal junction. Histologically, and by culture, the number of bacilli found in the liquefied caseum varied from many to comparatively few. Strong tuberculin reactions at 4 weeks after infection were associated with fewer primary lesions, while strong tuberculin reactions at 33 weeks were associated with more cavitary lesions. In the tuberculous granulation tissue surrounding caseous and liquefied pulmonary foci and cavities, we found many mature epithelioid macrophages that contained high levels of the proteinase cathepsin D. Therefore, cathepsin D probably plays a major role in the liquefaction of solid caseous material and in the subsequent cavity formation.

Nordihydroguaiaretic acid blocks IL-2-independent lymphocyte proliferation and enhances responses to PPD.

The authors examined the mechanism by which the non-specific lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibits human lymphocyte proliferative responses and compared its effects with those of cyclosporine (CsA). It was found that CsA-resistant proliferative responses induced by direct PK-C activators such as mitogenic concentrations of the phorbol ester TPA or the putative antitumour agent bryostatin 1 (bryo 1) were inhibited in a concentration-dependent manner by NDGA (IC50 = 2 microM). In contrast, CsA-sensitive IL-2-dependent proliferative responses induced by PHA, anti-CD3 or the purified protein derivative (PPD) of M. tuberculosis were not significantly inhibited by NDGA concentrations as high as 8 microM. The expression of the IL-2R by lymphocytes was also resistant to NDGA concentrations that effectively blocked the mitogenic effects of TPA or bryostatin, but could be inhibited by higher concentrations of NDGA (IC50 = 8 microM). In addition, NDGA, but not CsA, blocked the production of IL-6 by human mononuclear cells. Furthermore, PPD-induced proliferation was significantly enhanced by NDGA. These data would suggest that NDGA at concentrations below 8 microM selectively inhibits IL-2-independent proliferation. NDGA's ability to inhibit IL-6 while enhancing the proliferative response to PPD may indicate an anti-inflammatory therapeutic potential of antioxidants in mycobacterial infections.

Infection of SCID mice with Mycobacterium leprae and control with antigen-activated "immune" human peripheral blood mononuclear cells.

The SCID (severe combined immunodeficient) mouse lacks both B and T cells and tolerates injected mononuclear cells from humans, the principal hosts of Mycobacterium leprae. A SCID mouse model of leprosy could be useful to investigate potential vaccine strategies using human cells in a context in which the growth of the organism is monitored. Initial experiments determined that SCID mice are more susceptible than normal mice to infection and dissemination of M. leprae. Cells from humans, either BCG vaccinated or from countries where leprosy is endemic, were stimulated in vitro with a number of mycobacterial antigens--whole M. leprae, M. leprae cell walls, purified protein derivative of M. tuberculosis, and Mycobacterium bovis BCG--and tested for proliferation and production of interleukin-6, tumor necrosis factor alpha, and gamma interferon. Cell walls were the most efficient and consistent in inducing all of these activities. In vitro-activated human cells retain function better after injection into SCID mice than nonactivated cells. To test the ability of cells to affect the growth of M. leprae in the footpads of SCID mice, cells from a known responder to mycobacterial antigens and from a nonresponder were activated by M. leprae cell wall antigens. The cells were harvested and coinjected with fresh M. leprae into the right hind footpads of SCID mice. After 3 months, there was no growth of M. leprae in the footpads of mice coinjected with cells from the mycobacterial antigen responder, while growth was uninhibited in mice receiving cells from the nonresponder. Future experiments will determine requirements for antigen specificity in inhibiting M. leprae multiplication.

Immune responses to fractionated cytomegalovirus (CMV) antigens after HIV infection. Loss of cellular and humoral reactivity to antigens recognized by HIV-, CMV+ individuals.

In order to delineate the molecular pathogenesis of the increased susceptibility to CMV disease in HIV infection, the patterns of antigen responsiveness in HIV-infected and non-infected individuals were investigated. CMV was fractionated by SDS-PAGE and electroblotted onto nitrocellulose. Lymphoproliferative responses of healthy HIV-, CMV+ individuals and HIV+, CMV+ asymptomatic patients to a whole CMV antigen preparation and to 20 fractions of nitrocellulose-bound CMV were then compared. Three fractions of approximate molecular weight of 130-165, 65-75, and 55-65 kD appeared to contain the major T cell stimulating antigens for HIV-, CMV+ individuals. A statistically significant depression of responses to fractions containing antigens in the ranges of 130-165 kD and 55-65 kD but not to whole CMV was seen in HIV+ individuals compared with controls. In healthy controls, the sum of the proliferative responses as measured by 3H-thymidine uptake to these three major fractions was approximately equal to the response to a whole CMV antigen preparation, whereas it was less than half of this response in five out of six HIV+ subjects. When antibody activities to CMV antigens were analysed by immunoblotting of sera from the two subject groups and also sera of ARC and AIDS patients, a selective loss of reactivity was revealed in 10 out of 19 HIV+ subjects to a band of 26-28 kD whereas all 15 HIV-, CMV+ controls recognized this band. Serum IgG and IgM values were both significantly higher in HIV+ individuals than in controls. These findings suggest that specific lesions in the repertoire of immune responsiveness to CMV antigens occur in HIV+ individuals.

T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components.

Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. lepare-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (Mr) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. less than 70 kDa) Mr range with a peak in the 10-25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower Mr. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher Mr range, in particular greater than 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.

Cellular, humoral, and gamma interferon responses to Mycobacterium leprae and BCG antigens in healthy individuals exposed to leprosy.

Protective immunity against mycobacteria is dependent on antigen-specific T cells. The antibodies induced upon immunization with mycobacteria have no apparent role in host protection. Serological techniques have detected some antigens that are also recognized by human T cells but may fail to recognize others. Potentially, there may be differences in the epitopes seen by the T and B cell anti-mycobacterial antigen repertoires. We have screened the different components of sonicated BCG or Mycobacterium leprae that were separated according to their molecular weight (MW) by SDS-PAGE and then electroblotted on nitrocellulose paper. The blots were cut into squares and tested directly in a T cell proliferation assay. Our results indicate that peripheral T cells of healthy leprosy patient contacts respond preferentially to the lower MW (less than 70,000) and not the higher MW fractions of M. leprae and BCG, in contrast to the humoral response of these same individuals. The most important fractions in inducing a lymphoproliferative response were in the regions of 11-16 kDa of BCG and M. leprae and to the 22-26 kDa region of M. leprae. These fractions appeared to represent molecular weight regions that were in some instances clearly distinct from previously defined antigens. It was further shown that lymphoproliferation in response to mycobacterial fractions correlated with the production of gamma interferon, a lymphokine required for macrophage activation and elimination of mycobacteria. These studies allow the direct assessment of antigens involved in protective T cell-mediated immunity, and should be helpful in selecting relevant antigens for skin testing and immunization.

HLA-DR and DQ antigens in insulin-dependent diabetics in Ethiopia.

Thirty-one Ethiopian insulin-dependent (or type I) diabetes mellitus (IDDM) patients and thirty-three healthy controls from the same ethnic background were typed for HLA-A, B, C, DR and DQ specificities. The frequencies of both DR3 and DR4 were significantly increased among IDDM patients (resp. p = 0.02, p = 0.01), confirming results in other populations. In contrast to observations in Caucasians, no significant negative association was found with TA10, a newly recognized DQ specificity, at least in the population studied here, whereas DQwl was more frequently observed among healthy controls (p = 0.01). Although this latter difference does not retain statistical significance after correction for the number of comparisons made, these findings may support previous results suggesting the existence of IDDM susceptibility genes associated with DR3 and DR4 and of IDDM resistance genes associated with DQ antigens.

HLA antigens and neural reversal reactions in Ethiopian borderline tuberculoid leprosy patients.

Reversal reactions (RR) or acute neuritis episodes are frequently observed in borderline tuberculoid (BT) leprosy patients during the first year of treatment, and are associated with a rapid increase in cell-mediated immunity. Because HLA-linked genes have been shown to be an important factor in determining the type of leprosy that develops in susceptible individuals and because HLA molecules regulate cellular interactions in the immune system, we have investigated whether RR are associated with HLA antigens in Ethiopian patients. The data reported here indicate that this is not the case: no significant differences in the distribution of HLA class I and class II antigens were observed among three groups: 28 BT patients with a history of RR, 27 BT patients with no history of RR, and 33 healthy individuals. In contrast to these negative results, we observed that HLA-DR3 was associated with high skin-test responsiveness against Mycobacterium leprae antigens among RR patients. Since DR3 was not associated with RR per se, the observed DR3-associated high responsiveness to M. leprae may not be primarily related to the development of RR.

Cyclosporine inhibits soluble antigen and alloantigen presentation by human monocytes in vitro.

We examined whether cyclosporine (CsA) interferes with human monocyte processing and presentation of soluble and particulate antigens. Human monocytes were pulsed in the presence of CsA with one of three antigens: tetanus toxoid (TT), diphtheria toxoid (DIP) or cytomegalovirus (CMVx). When these monocytes were co-cultured with immunocompetent T-lymphocytes they were found to be impaired in the induction of proliferative and cytotoxic T-cell responses. At CsA concentrations higher that 0.5 micrograms/ml, proliferation induced by the particulate CMVx antigen was markedly more sensitive than that induced by the soluble TT antigen. In the allogeneic mixed lymphocyte reaction (MLR), pretreatment of responder haplotype monocytes alone with CsA led to reduced reactivity as determined in both proliferative (P less than 0.01) and cell-mediated lympholysis (P less than 0.001) assays. These effects of CsA were not due to direct CsA action of residual CsA carried over with treated monocytes, and were not apparently due to inhibition of interleukin-1 (IL-1) production since exogenous IL-1 could not restore normal responses.

Natural killer (NK) cell activity and reversal reaction in leprosy.

Two studies were conducted to assess natural killer (NK) cell activity in leprosy patients and healthy Ethiopian controls. The first study tested 26 untreated leprosy patients across the spectrum of the disease. It was found that lepromatous leprosy and all untreated, nonreactional patients had lower NK activity than healthy controls. However, patients presenting with reversal reaction (RR) had NK activity within the normal range. Heterogeneity was particularly marked in the NK activity of borderline patients. In the second study, NK cell activity was assessed in treated borderline tuberculoid leprosy (BT) patients. There were 30 patients with a history of RR and 27 BT patients without such a history (NR). All patients had had at least 3 years of dapsone treatment and 6 months of multidrug therapy. There were 26 control subjects. NK activity was higher in controls than in patients only at one effector:target (E:T) ratio tested, but NK cells from the BT patient group appeared to be more "aggressive" in that there was significantly (p less than 0.001) less reduction of activity with dilution of effector cells. There were no significant differences in NK activity between RR and NR patients. The NK activity of NR patients was positively correlated with the size of induration of the lepromin response. We conclude that higher NK activity in acute RR would appear to be a consequence rather than a cause of reversal reactions.

An in vitro predictive test for graft versus host disease in patients with genotypic HLA-identical bone marrow transplants.

Acute graft versus host disease remains a major cause of morbidity and mortality in allogeneic bone marrow transplantation. To date, no clinically useful test has been reported that will predict the occurrence of graft versus host disease in genotypic HLA-identical donor-recipient pairs. We have developed a skin-explant model using donor lymphocytes that have been sensitized against recipient lymphocytes in vitro and cocultured with the recipient's skin. Histologic changes compatible with acute graft versus host disease are found in the positive explants. To date 32 patients have been tested in a prospective manner. Among the 18 recipient-donor pairs that were positive, 16 patients were found to have histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Among the 14 negative pairs, only 3 patients had histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Thus, the model has a sensitivity of 84 per cent and a specificity of 85 per cent, and is a significant predictor of the histologic occurrence of graft versus host disease (P less than 0.0005 by chi-square test). The test may be useful in the selection of donors for bone marrow transplantation and in the planning of prophylaxis against graft versus host disease.

Effect of cyclosporin and interleukin-2 on the restoration of in vitro immune responses to cytomegalovirus.

Previous studies have shown that cyclosporin (CSA) inhibits lymphoproliferation to cytomegalovirus (CMV)-infected, glutaraldehyde-fixed, and irradiated fibroblasts (CMVFFx) in vitro. Generation of cytotoxic cell activity is impaired in cultures with CSA, but the induction of suppressor cells is not. In the present studies we tested the ability of interleukin-2 (IL-2) and supernatants of lymphocytes stimulated by CMVFFx with or without CSA (1 microgram/ml) to restore functional activities of lymphocytes from primary cultures treated or not treated with CSA. IL-2 significantly enhanced lymphoproliferation, cell-mediated cytotoxicity to CMV-infected fibroblasts (CMVF), natural killer cell activity, and the activity of cells capable of suppressing the response of fresh autologous cells to CMVFFx of cells derived from control and CSA-treated primary cultures. IL-2 was found in day-2 supernatants of control cultures but not CSA-treated cultures. Day-2 control supernatants were capable of significantly enhancing proliferation and suppressor cell activity but were less efficient at restoring cytotoxic cell function. Day-2 supernatants from CSA-treated cultures were not able to enhance lymphoproliferation or cytotoxic cell function but did induce significant levels of suppressor cell activity. The results indicate the presence of different functional mediators in the culture supernatants. The ability of IL-2 to restore lymphocyte effector functions against a clinically important virus may have important therapeutic implications in the treatment of this viral infection in immunodeficiency diseases and in the restoration of immune competence after transplantation.

Effect of cyclosporine on the response of normal human lymphocytes to cytomegalovirus in vitro.

Lymphocytes from healthy volunteers seropositive for cytomegalovirus (CMV) demonstrated strong lymphoproliferative responses to CMV-infected and glutaraldehyde-fixed foreskin fibroblasts (CMVFFx) when cocultured for 6 days. Addition of the immunosuppressive drug cyclosporine (CsA) to the cultures resulted in a 10-fold reduction (P less than 0.001) in counts per minute of [3H]thymidine uptake. The proliferative response to noninfected fixed fibroblasts was also reduced 10-fold. Kinetic studies showed an inhibition of the lymphoproliferative response and not an alteration in the time course kinetics in CsA-treated cultures. Cytotoxicity to CMV-infected and unfixed fibroblasts by lymphocytes primed by CMVFFx in the presence of 0.5 micrograms of CsA per ml was significantly reduced (P less than 0.01) as compared with untreated cultures but remained significantly above background level (P less than 0.01). The cytotoxic response was still present but reduced at concentrations of greater than or equal to 1.0 micrograms/ml. Cytotoxicity to noninfected fibroblasts by CMVFFx-primed lymphocytes could be eliminated by as little as 0.5 micrograms of CsA per ml. Stimulation of lymphocytes by CMVFFx but not by noninfected fixed fibroblasts resulted in the in vitro generation of suppressor cells. CsA at a final concentration of 1.0 or 2.5 micrograms/ml did not significantly impair the induction of cells capable of suppressing the lymphoproliferative response of fresh autologous mononuclear cells to CMVFFx. The findings described above may have important clinical implications in that a degree of protective immunity to CMV-infected cells is maintained even in the presence of CsA.