Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation.

Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3-4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 microM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 microM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.

Immunologic monitoring with Orthoclone OKT3 therapy.

Muromonab-CD3 monoclonal antibody (Orthoclone OKT3) was used 146 times in 123 transplant recipients to treat or prevent rejection. Reversal and prevention of rejection were evaluated 1 week and 1 year after OKT3 therapy. Eighty-one percent (73 of 90) of the rejection episodes in kidney transplant patients were reversed with 67% of these grafts functioning at 1 year. Eighteen of 20 (90%) rejection episodes in liver transplant recipients were reversed, as were 11 of 13 (85%) heart transplant rejection episodes. Only one of five pancreas transplant episodes were reversed. OKT3 was used prophylactically in 18 transplant recipients (13 kidney, four heart, one liver). Immunologic monitoring (lymphocyte subsets, serum OKT3 levels, and antimurine antibodies) was performed during and after OKT3 therapy. Antimurine antibody formation rate was 28% (26 of 94 patients monitored). OKT3 therapy resulted in a rapid depletion of CD3+ cells from the peripheral circulation (less than 20/mm3) and trough serum OKT3 levels of greater than 800 ng/ml by the third day of therapy in all transplant types. Twenty-three patients (14 kidney, five liver, three heart, and one pancreas) were retreated with OKT3; reversal of rejection occurred in 87% of patients (13 of 15) with no antimurine antibodies and in 83% of patients (five of six) with a low antibody titer but did not occur in the two patients with a high antibody titer. Retreatment of patients with no anti-OKT3 antibody resulted in a depletion of CD3+ cells from the peripheral blood, but it took longer than in patients treated with OKT3 for the first time. Similarly, serum OKT3 levels increased slower in retreated patients compared with first treatment. In retreatment patients with a low titer antimurine antibody, often it was necessary to increase the dose of OKT3 to achieve adequate serum OKT3 levels and to deplete CD3+ cells. Antimurine antibody developed de novo in four of the 15 antibody negative patients (27%) who were retreated. Overall, OKT3 was an effective agent in reversing and preventing rejection in solid organ transplantation with few severe side effects and a low mortality. Retreatment with OKT3 should not be considered unless the antibody status of the patient is known. Development of low titer antibodies does not preclude successful retreatment with OKT3. Alternate antirejection therapy, however, should be used in patients with high titer antimurine responses.

Influence of operations with cardiopulmonary bypass on polymorphonuclear leukocyte function in infants.

To determine the effect of operations with cardiopulmonary bypass on the immunologic function of polymorphonuclear leukocytes in infants, we studied polymorphonuclear leukocyte function and immunologic profile in 16 infants undergoing repair of congenital heart lesions. An oxygen/air/high-dose fentanyl anesthetic was used for all patients. Absolute neutrophil count increased significantly (p less than 0.05) after bypass and remained increased 48 hours afterward. Chemotaxis, random migration of polymorphonuclear leukocytes, and phagocytic index were unaffected, but bactericidal capacity decreased significantly immediately after cardiopulmonary bypass and remained decreased 48 hours later. Serum opsonizing capacity to bacterial and fungal antigens was variably altered, and complement factors 3 and 4 decreased significantly after cardiopulmonary bypass. Total hemolytic complement decreased significantly immediately after cardiopulmonary bypass and returned to normal by 48 hours. These data suggest that operations with cardiopulmonary bypass in infants significantly affect the immunologic function of polymorphonuclear leukocytes and result in consumption of complement.

Age-dependent variation of lymphocyte function in the postoperative child.

Circulating lymphocyte profiles and reactivity normally vary with age. Operation results in depression of both lymphocyte counts and blastogenesis but the relationship of age to these alterations has not been previously evaluated in the pediatric surgical patient. This report analyzes the relationship of age to lymphocyte alteration in the postoperative child. Thirty-five healthy children (age range 1 mo to 12 yr), admitted for elective herniorrhaphy, underwent perioperative lymphocyte assay. Anesthesia consisted of halothane and NO2, and operative time averaged 54 minutes. Three milliliters of heparinized whole blood was obtained at induction of anesthesia and at 2 hours postoperatively. Lymphocytes were separated by Histopaque gradient and assayed for absolute count, total T-cells, and total B-cells. Lymphocyte reactivity was determined by 3H-Thymidine incorporation during incubation with the mitogens phytohemagglutinin (PHA), pokeweed (PWM), and/or concanavalin A (Con A) and results expressed as the logarithm of scintillation counts per minute. Differences in preoperative and postoperative values were analyzed for significance by paired T-test. The same differences were evaluated for relationship to age by regression analysis. Operation resulted in significant decreases in all lymphocyte counts (absolute, T- and B-cells), and the operative-induced alteration in both absolute and total B-cell counts were significantly correlated with age (P less than .03, P less than .007, respectively). Similarly, operation resulted in significant depression of lymphocyte reactivity and alteration in B-cell response (PWM) was significantly correlated with age (P less than .008). In all instances of significant correlation, lymphocyte alterations were inversely related to age.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte survival in cerebrospinal fluid.

Delays in the laboratory examination of cerebrospinal fluid are commonly encountered in clinical medicine. The present studies were designed to evaluate changes in cerebrospinal fluid leukocyte counts relative to time elapsed before analysis. Neutrophil counts decreased most rapidly, being 68 +/- 10% (standard error of the mean) and 50 +/- 12% of initial values at 1 and 2 h, respectively. Lymphocyte and monocyte numbers were not significantly altered until 3 h.

Immunologic responses following serial skin testing.

Twenty volunteers underwent intradermal skin testing with tetanus toxoid (TT) and varicella-zoster (VZ) antigens weekly for 7 weeks. In addition to measurements of delayed hypersensitivity skin test responses, in vitro antigen specific lymphocyte stimulation and antibody levels were monitored. Data for TT exhibited more variability than VZ, demonstrating a marked boost in antibody titers after the first skin test and a shift in the kinetics of the skin test reaction to later maximum responses after the first 3 weeks. In contrast, serial skin testing with VZ was not associated with changes in cellular immune assays. Following the initial VZ skin test there was a slight rise in antibody levels for one-third of studied individuals. These observations suggest that VZ skin test antigen is ideal for monitoring immune function and that repeated testing should not significantly influence in vitro assays.

Immunotoxicology of brown recluse spider (Loxosceles reclusa) venom.

A purified toxic protein from Loxosceles reclusa venom was assayed for its in vitro effects on the human immunological and blood clotting systems. The toxin caused inhibition of neutrophil chemotaxis, depletion of serum hemolytic complement, prolongation of the activated partial thromboplastin time and depletion of clotting factors XII, XI, IX and VIII by an average of 44% in human plasma. The prothrombin time of human plasma was also prolonged by 1.5-2.0 sec. No effect of the purified toxin was observed on microbicidal ability of neutrophils, the release of enzymes from neutrophils or the adherence of neutrophils to glass beads.

Psychologic modulation of the human immune response to varicella zoster.

Psychoimmunology, the interrelationship between the brain/mind/psyche and the immune system, is now an established area of scientific research. Based on prior investigations we hypothesized that an experienced meditator could affect her delayed hypersensitivity reaction by a psychological process. A single-case study design was employed in which the subject was skin tested weekly with varicella zoster skin test reagent. After baseline immunologic studies, she was able, as hypothesized, to significantly reduce both the induration of her delayed hypersensitivity skin test reaction and in vitro lymphocyte stimulation to varicella zoster. Then, as predicted, she was able to allow her reaction to return to baseline. As a confirmation of what is to our knowledge this previously undescribed phenomenon, she was able to reproduce the entire sequence nine months later. It appears that this subject can intentionally modulate her immune response by a psychologic mechanism.

Lymphocyte subpopulations of blood and alveolar lavage in blastomycosis.

Patients with blastomycosis were found to have differing lymphocyte populations depending on the extent of disease manifestations and whether or not therapy had been started. Patients with recovering pulmonary blastomycosis who had been receiving antifungal treatment for at least four weeks had lymphocyte subpopulations no different from control donors. Patients with treated extrapulmonary blastomycosis had similar T helper (TH) to T suppressor (TS) ratios compared to recovering pulmonary patients and control subjects; this ratio gives a false impression because extrapulmonary blastomycosis patients had a reduced absolute number of lymphocytes with either marker. In bronchoalveolar lavage fluid, pulmonary blastomycosis patients who were clinically improved while receiving antifungal therapy had fewer TH cells and a greater number of lymphocytes with the TS marker than did control subjects. Patients with pulmonary blastomycosis prior to therapy had a smaller TH/S ratio than the other groups in peripheral blood primarily due to a reduction in the circulating TH fraction in both absolute numbers of cells and in the percentage of total T lymphocytes. Pulmonary blastomycosis patients re-evaluated after at least four weeks of antifungal therapy had TH/S ratios that were similar to normal persons. This increase in TH lymphocytes corresponded to clinical improvement and in a temporal correlation to that described for the development of specific immunity in this illness.

Intravenous immune globulin for hypogammaglobulinemia: a comparison of opsonizing capacity in recipient sera.

Twelve severely hypogammaglobulinemic patients received infusions of alkylated immune globulin and two other native nonalkylated products. Administration was separated by an interval of 3 weeks. Serum was obtained prior to and at 24 hr and 3 weeks after each infusion for measurement of total IgG, specific and opsonizing antibodies. The latter was accomplished against Streptococcus pneumoniae types 5, 12F and 14 and zymosan using chemiluminescence methodology. Changes in total IgG concentrations were comparable for the three products. Prior to enrollment, IgG levels averaged 115 +/- 72 mg/dl, increasing to 779 +/- 399 at 24 hr postinfusion, and were 337 +/- 200 after 3 weeks. No differences among the products were seen in their ability to produce antibodies against Herpes simplex virus types 1 and 2, rubella, toxoplasma, cytomegalovirus, or tetanus. However, differences in opsonizing antibody were observed between alkylated and native IgG preparations. Peak chemiluminescence responses of neutrophils following opsonization of S. pneumoniae with native immune globulin were significantly higher than with alkylated IgG, indicating greater functional capacity. These studies suggest that native immune serum globulin provides a greater potential for augmenting host defense mechanisms against pneumococcal infection in hypogammaglobulinemic patients.

Intracellular growth and phagocytosis of Blastomyces dermatitidis by monocyte-derived macrophages from previously infected and normal subjects.

Blastomyces dermatitidis evokes responses of human cellular immunity typical of other intracellular fungal pathogens. Differences in growth rates of intracellular Blastomyces yeast and the differences in amounts of yeast phagocytized by macrophages were determined for macrophages derived from peripheral blood monocytes from 11 persons with treated blastomycosis and 11 normal, healthy persons. Cellular immunity was examined by lymphocyte uptake of [3H]thymidine in response to a specific antigen of Blastomyces yeast. Yeast were more readily phagocytized by macrophages from the previously treated donors when compared with those from the normal donors; the yeast were confirmed to be intracellular by transmission electron microscopy. Likewise, a decrease in growth rates of yeast was demonstrable in cultures of macrophages from previously treated donors as compared with normal donors. This greater efficiency of phagocytosis and growth inhibition of B. dermatitidis reflects another mechanism of human cellular immunity to this fungal infection.

Surgically induced immunologic alterations in the child.

Surgery is generally believed to be an immunodepressant. This assumption is based, in part, upon studies of compromised patients undergoing major operation. Similar studies in normal adults following elective procedures are contradictory and little information is available regarding the pediatric surgical patient. This paper presents a study of immune function in children undergoing elective operation. Fifty healthy preoperative children (mean age: 20 months) were randomly selected. Ninety-five percent underwent inguinal herniorrhaphy. Operative time averaged 45 minutes (range: 30 to 90 minutes). Anesthesia consisted of Halothane and Nitrous Oxide in all cases. Approximately 2.5 cc of heparinized blood and 0.5 cc of serum were obtained immediately prior to and 2 hours following operation. Half of the children underwent assays of neutrophil function including absolute count, random migration, chemotaxis, phagocytosis, and bacterial killing. Serum was examined for opsonization of Staphylococcus aureus using the chemiluminescence method. The remaining children underwent lymphocytic quantitation including absolute count, total T cells, total B cells, T-helper cells, T-suppressor cells, and T-helper/suppressor ratio. Absolute neutrophil count increased 2.4 times preoperative values (P less than 0.01). There were, however, no significant alterations in neutrophil functional capabilities. Similarly, there was no alteration in serum opsonic capacity. There was a significant decrease in absolute lymphocyte count (6560-4013, P less than 0.01) postoperatively, and T cells, T-helper, T-suppressor, and B cells were all significantly affected (P less than 0.01 to 0.02). There was no change in the T-helper/suppressor ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic aspects of human endometriosis.

General and specific immune function were examined in women with endometriosis. Nonspecific parameters included total leukocyte and differential counts, quantitative immunoglobulin (IgG, IgA, and IgM) determinations, total hemolytic complement, C3, C4, mitogen-induced lymphocyte stimulation, and human leukocyte antigen (HLA) typing; no differences were observed when data were compared to age-matched controls without endometriosis. In contrast, the specific immune response T-lymphocyte-mediated cytotoxicity to autologous endometrial cells was significantly reduced (P less than 0.01) in women with endometriosis. When results from patients were analyzed according to clinical severity of endometriosis, even more striking immunologic alterations were delineated. In addition, lymphocyte stimulation responses to autologous endometrial antigen were lower in patients with severe or moderate disease, approaching statistical significance (P = 0.18 and 0.12, respectively). These studies suggest an immunologic basis for development of endometriosis.

Comparison of flow cytometric analysis and [3H]thymidine incorporation for measurement of the effects of drug toxicity on lymphocyte stimulation.

Human mononuclear cells were exposed to three antiviral agents, stimulated with phytohemagglutinin, and assayed for DNA synthesis with [3H]thymidine uptake and flow cytometric analysis. Cytosine-arabinoside demonstrated inhibition of blastogenic reactivity by both [3H]thymidine uptake and flow cytometric analysis, whereas acyclovir showed no significant suppression. In contrast, (E)-5-(2-bromovinyl)-2'-deoxyuridine, a thymidine analog, demonstrated a lack of correlation between the two methods. The competitive inhibition of some compounds with [3H]thymidine incorporation necessitates the use of other methods to measure DNA synthesis.

Fatty acids and the inhibition of mitogen-induced lymphocyte transformation by leukemic serum.

The effects of sera from 23 patients with acute lymphocytic leukemia on mitogen-induced transformation of normal human lymphocytes were examined. All sera (100%) at diagnosis and 70% of those obtained during the induction of remission demonstrated inhibition of mitogen-induced lymphocyte transformation as evidenced by decreased uptake of [3H]thymidine. The inhibition could not be overcome by an increase in the mitogen concentration. Eighteen sera demonstrating a mean inhibition of 44.2% had elevated levels of free fatty acids (FFA) compared with five sera showing no inhibition (p value = 0.001). Inhibition, within the range produced by leukemic sera, could be achieved by the purified methyl esters of linoleic and linolenic acid in this system. Flow cytometric analysis of mitogen-induced cycling of normal human lymphocytes suggested that deoxyribonucleic acid (DNA) replication was inhibited by leukemic sera containing elevated levels of FFA after the G1 phase of cell cycling. Similar inhibition could be achieved by the methyl ester of linolenic acid in this system. Gas chromatographic analysis revealed that sera demonstrating inhibition contained linolenic (C18:3) acid (p value = 0.015), and the majority showed one or more of the following: 1) a concentration of oleic (C18:1) acid greater than 2 standard deviations (SD) above the mean of the control leukemic sera (i.e., those not demonstrating inhibition); 2) an arachidonic (C20:4) to C18:1 ratio that is reduced to greater than 2 SD below the mean of the control group; or 3) the presence of an unexpected fatty acid fraction. The data raise the question of an influence of FFA and, specifically, an interplay of unsaturated fatty acids on immune function during the natural history of acute lymphocytic leukemia.

Cellular immunotoxicity of amyl nitrite.

Functional deficits in lymphocyte interaction following occasional or chronic exposure to inhaled nitrites may be a potential contributing but not the etiologic factor in the acquired immunodeficiency syndrome (AIDS). We evaluated the effect of amyl nitrite vapors on mononuclear cell function and demonstrated functional deficits and structural alterations in these cells. In this closed, in vitro system, exposure of cells to amyl nitrite for up to 30 minutes did not effect cell viability. The functional deficits demonstrated were: inhibition of lymphocyte erythrocyte (E) rosette formation, a suppression of lymphocyte mitogen (phytohemagglutinin) and antigen (cytomegalovirus) transformation, a block in the S, G2 and M phases of cell cycling and diminished cell cytotoxicity to CMV infected cells. These effects on cellular function were demonstrated following 5, 10, and 15 minutes of amyl nitrite vapor exposure; some effect on all cellular functions was demonstrated at 5 minutes. The structural alterations seen on scanning and transmission electron micrographs were: reduction of filapodia, smoothing of the cell profile, cytoplasmic protrusions with pseudopod-like extensions, an increase in rough endoplasmic reticulum with swollen cisternae, alterations in size and distribution of golgi components and exocytotic vesicles in the outer membrane of the nuclear envelope. These vesicles and increased membrane proliferation suggests an effect on the membrane synthesis mechanism in these cells. These effects may be a potential factor in the alterations of phenotypic markers on T lymphocyte populations, as well as, a potential contributing factor in the functional deficit of mononuclear cells in patients with AIDS.

Functional bacterial opsonic activity of human amniotic fluid.

There are some data to suggest that amniotic fluid protects the fetus from invasion by pathogenic bacteria. To examine methods by which amniotic fluid may offer such protection, quantitative antibody, complement activity, and functional opsonic capacity were measured. Immunoglobulins were measured by laser nephelometry and total hemolytic complement was determined by radial diffusion; results suggested activity adequate for bactericidal capacity. The chemiluminescence assay was used to quantitate the functional interaction between polymorphonuclear leukocytes and E. coli, group B streptococci (GBS), or zymosan particles preopsonized with amniotic fluid obtained at different stages of gestation. Results were compared to those for normal serum. Data were analyzed by evaluation of the initial slope, area under the curve, and peak chemiluminescence response. Opsonic activity of amniotic fluid for E. coli and GBS was demonstrated, with E. coli showing greater reactivity (maximum = 15,000 to 25,000 cpm) than GBS (10,000 to 20,000 cpm). Specific, as well as nonspecific, opsonic activity was demonstrated by absorption of amniotic fluid with killed bacteria. Concentration of amniotic fluid did not result in an increase in chemiluminescent activity, which demonstrates that optimal opsonic activity already exists. The classical and alternate pathways of complement were assessed for E. coli and GBS. Preterm amniotic fluid did not differ in response from that of amniotic fluid obtained from term pregnancies. This study demonstrates that amniotic fluid can provide the fetus with protection from bacterial pathogens and delineates mechanisms for such protection.

Immune responses to varicella-zoster in the aged.

Skin test reactivity and in vitro lymphocyte stimulation responses to varicella-zoster (VZ) were examined in a large normal population ranging in age from 6 months to 93 years. Warning of cellular immunity, as examined by skin delayed hypersensitivity, began at age 40 years. Skin test responses to phytohemagglutinin, however, remained positive into the eighth decade of life. In vitro lymphocyte stimulation responses to VZ were usually positive (stimulation index greater than or equal to 2.5) until age 60 years, after which time levels, as observed with nonimmune individuals, were often demonstrated. Antibody levels, as measured by fluorescent antibody to membrane antigen, remained positive into the ninth and tenth decades of life. This was especially so with a history of reactivation (zoster) VZ infections, while skin test and in vitro responses were rarely positive in those individuals. This cellular, as contrasted with humoral, immunity decreases with advancing age, which may account for a propensity to reactivation of VZ virus.

Ketoconazole, amphotericin B, and amphotericin B methyl ester: comparative in vitro and in vivo toxicological effects on neutrophil function.

We investigated a number of parameters for host defense after the in vitro addition of the antifungal agents ketoconazole, amphotericin B (AMB), and amphotericin B methyl ester (AME). Similar assays were repeated before and after patients received the former two drugs. Viability by trypan blue exclusion, adherence by nylon wool columns, chemotaxis by the under-agarose technique, phagocytosis and killing by chemiluminescence, colony counts, and acridine orange direct visualization were assayed. In striking contrast to AMB and AME, ketoconazole demonstrated no significant effect on neutrophils. Adherence in the presence of therapeutic plasma levels of AMB and AME was decreased (P less than or equal to 0.005) at low drug concentrations, whereas at higher concentrations, adherence was increased (P less than 0.001). The chemotactic responses of cells incubated with AMB and AME demonstrated marked suppression. Phagocytic capacity and killing were decreased (P less than or equal to 0.005) with AMB as compared with control assays and assays performed in the presence of ketoconazole and AME. However, no difference were observed between two patients who received AMB and two other treated with ketoconazole.

Immune deficiency in fetal alcohol syndrome.

In a review of 13 documented cases of fetal alcohol syndrome (FAS), an increased incidence of life-threatening bacterial infections as well as a propensity to minor infections was observed. Five of 13 patients had had at least one episode of pneumonia, two had meningitis, and one had sepsis. A comprehensive immunologic evaluation of FAS was completed, and results were compared to an age-matched control group of children with intrauterine growth retardation without FAS. Children with FAS were shown to have decreased E rosette-forming lymphocytes (35 +/- 5% versus 55 +/- 5% or 1328 +/- 274 versus 2333 +/- 112 per mm3), low EAC rosette-forming lymphocytes (15 +/- 2% versus 18 +/- 1% or 524 +/- 109 versus 740 +/- 75 per mm3), and diminished mitogen-induced stimulation responses to mitogens: 31616 +/- 5337 versus 58076 +/- 4455 cpm for phytohemagglutinin, 17582 +/- 5436 versus 35018 +/- 5346 for pokeweed mitogen, and 32460 +/- 7044 versus 54996 +/- 5531 for concanavalin A, P less than 0.05. Nine patients had dysgammaglobulinemia, FAS subjects also had a marked eosinophilia (624 +/- 154 versus 72 +/- 27 mm3). Other parameters of immune function including absolute lymphocyte and neutrophil counts, total hemolytic complement, delayed cutaneous hypersensitivity, and nitroblue tetrazolium dye reduction assays, were not different from control children. Impairment of immunity may explain an increased susceptibility to infection in FAS.