J. George Bekesi

Increased expression of activation markers on CD8 lymphocytes in children with human immunodeficiency virus-1 infection.

ABSTRACT: The aims of the present study were to analyze the impact of perinatal human immunodeficiency virus (HIV)-1 infection on lymphocyte maturation in children, to determine the expression of activation markers on CD8+ cells, and to define predictors of survival in HIV-infected children. Seventy-one children presenting HIV-related symptoms were included in the study; 29 were less than 2 y old and 42 were 2 to 12 y of age. Results were compared with those obtained in normal children of a similar age. In HIV-infected children the proportion of CD4+ and CD8+ CD45RA+ cells was significantly decreased, whereas that of CD8+, CD8+CD38+, and CD8+CD45RO+ cells was strikingly increased compared with controls. In children less than 2 y old the absolute number of CD4+ and CD8+CD45RA+ cells decreased, and the number of CD8+CD45RO+ cells increased significantly, whereas the number of CD8+ and CD8+CD38+ cells did not change. The absolute number of CD4+ T cells declined with age both among controls and among HIV-infected children. In contrast, the absolute number of CD8+ cells and CD8 subsets decreased with age only in controls but not in infected children. In HIV-1-infected children the expression of the CD38 and CD45RO markers on CD8+ cells was significantly correlated, indicating that these were activated cells. The survival of less than 2-y-old children with AIDS symptoms was positively correlated with the total number of CD8 cells and CD8+CD38+ cells at time of entry into the study. Most of the children who died by the end of the study had a CD8 count of less than 750/mm3 and a CD38+CD8+ count of less than 600/mm3 when first seen, whereas most of those who were alive had higher counts.

Increases in soluble CD8 antigen in plasma, and CD8+ and CD8+CD38+ cells in human immunodeficiency virus type-1 infection

Increases in plasma levels of soluble CD8 (SCD8) antigen and expansion of the CD8+ CD38+ lymphocyte compartment were early immunologic alterations frequently observed prior to detection of antibodies against human immunodeficiency virus type 1 (HIV-1) and diminution of CD4+ cells in subjects at risk to develop AIDS. These increases identified in the 49 seronegative homosexual men were manifest in all 164 homosexual subjects and 45 intravenous drug users (IVDU) positive for HIV-1 antibodies (HIV-1+), 19 patients with ARC, and 29 AIDS patients. Augmentation of plasma sCD8 antigen correlated with increases in both CD8+ and CD8+ CD38+ cells in HIV-1(-) homosexual men (r = 0.35, P less than 0.013; r = 0.48, P less than 0.0005; respectively) and the 258 HIV-1+ subjects (r = 0.25, P less than 0.0003; r = 0.33, P less than 0.0001, respectively). In vitro examination of unstimulated peripheral blood lymphocytes from HIV-1+ homosexuals and IVDU confirmed the fivefold higher constitutive levels of cellular release of sCD8 antigen in these subjects compared to heterosexual controls. Inclusion of radiolabeled amino acids during the 3-day culture period in the presence or absence of phytohemagglutinin resulted in negligible levels of radioactivity associated with the sCD8 antigen indicative of a lack of de novo synthesis. Throughout clinical progression to AIDS, sCD8 antigen levels continued to escalate relative to the numbers of CD8+ cells bearing CD38+ antigen. The data confirm the interrelationship between sCD8+ antigen and CD8+ and CD8+ CD38+ cells.

Elimination of 2-deoxyribose interference in the thiobarbituric acid determination of N-acetylneuraminic acid in tumor cells by pH-dependent extraction with cyclohexanone.

Abstract The widely used thiobarbituric acid technique for the quantitation of N -acetylneuraminic acid (NANA) was improved to eliminate the interference of the ubiquitous 2-deoxyribose. The 2-deoxyribose chromogen was completely removed by cyclohexanone extraction at pH 5.6–6.0. After readjusting the pH to 1.7–1.9, the chromogen representative of NANA was quantitatively extracted with cyclohexanone. All other aspects of the original technique (L. Warren 1959 J. Biol. Chem. 534 , 1971–1975) remained unchanged. The technique has been applied to determine total as well as neuraminidase-susceptible NANA in the preparation of the immunogen (neuraminidase-treated myeloblasts) utilized to stimulate specific immunity in patients with myeloblastic leukemia and certain solid tumors; NANA levels significantly affect immunogenicity. Data obtained from a variety of tumors using pH-dependent extraction as compared to the thiobarbituric acid method, isoamyl alcohol extraction, and ion-exchange purification showed that 2-deoxyribose interference may cause as much a two- to threefold error in the quantitation of NANA.

Impairments in functional subsets of T-suppressor (CD8) lymphocytes, monocytes, and natural killer cells among asbestos-exposed workers.

Peripheral blood leukocytes from asbestos-exposed workers were analyzed by dual color flow cytometry using monoclonal antibodies that identify developmental (HLA-DR) and functional (Leu 8) subsets of T helper, suppressor lymphocytes, and monocytes. An increase in the number of T suppressor cells was closely associated with a decrease in T lymphocyte functions while numerical defects in activated monocytes (Leu M3+Ia+) and natural killer cells (Leu 7+) were correlated with a depressed Th/Ts ratio. Furthermore, among asbestos-exposed workers with depressed T cell functions we have demonstrated a significantly higher number of the effector Ts (Leu 2+ Leu 8-) subset which regulates both the Th/Ts lymphocyte system as well as B cells and NK cell activities. These findings identified changes in the T suppressor feedback regulatory loop as being responsible for the immunoregulatory imbalance among long-term asbestos workers. In double blind analyses of demographic and radiographic data these phenotypic changes were not correlated with age, smoking history, or duration of exposure but were associated with radiographic evidence of asbestos-associated effects. This correlation established a direct link between asbestos exposure and the subsequent development of immune dysfunction.

A double-blind clinical trial of the effects of inosine pranobex in immunodepressed patients with prolonged generalized lymphadenopathy.

In a double-blind clinical trial, 61 immunodepressed males with persistent generalized lymphadenopathy (PGL) received one of two doses (1 or 3 g/day) of the immunomodulating drug inosine pranobex (INPX) or placebo for a period of 28 days. In the high-dose group, clinical improvement was reported by 11 of 21 patients (52%), within 5 months of the cessation of treatment. In contrast, 3 of 19 patients (16%) in the placebo group reported clinical improvement by that time. Patients receiving 3 g/day INPX showed a significant increase in NK cell activity by Day 14 and this elevation was still evident at the last follow-up examination 1 year after treatment. Increases in total T lymphocytes (T-11) and the percentage of T helper cells (T-4) were also observed. These responses were delayed and reached their peaks 2 months after the termination of drug treatment. The kinetics of these effects suggest that INPX stimulates the production of precursor cells and initiates a cascade of lymphocyte differentiation capable of producing long-term restoration of cell-mediated immunity. These data indicate that INPX may be beneficial to patients with PGL.

Isoprinosine-induced modulation of T-helper-cell subsets and antigen-presenting monocytes (Leu M3+ Ia+) resulted in improvement of T- and B-lymphocyte functions, in vitro in ARC and AIDS patients

Peripheral blood leukocytes from ARC and AIDS patients were analyzed following phytohemagglutinin- and pokeweed mitogen-induced lymphocyte transformation by dual-color flow cytometry using monoclonal antibodies that identify developmental (HLA-DR) and functional (Leu8) subsets of T cells and monocytes. Significant decreases in both the suppressor regulating helper T subset (Leu3+ Leu8+) and the reciprocal inducer helper T subset (Leu3+ Leu8-) responsible for inducing differentiation of B cells were observed. Simultaneously, the percentages of the effector suppressor T cells and the precursor suppressor T cells were increased, both of which were required for generation of suppression of cell-mediated immunity. There was also a progressive selection of Ia+ cells bearing the Leu2 (Ts) markers and a concurrent reduction of the percentage of antigen-presenting monocytes and activated helper T cells. These results suggest that the functional deficiencies in AIDS may be caused by defects in T-cell activation as well as antigen presentation by monocytes. Isoprinosine induced an increase in both regulator Th (Leu3+ Leu8+) and inducer Th (Leu3+ Leu8+) subsets of helper T cells while potentiating the expression of Ia antigen on helper T cells and monocytes during mitogen-driven DNA synthesis. These events initiated a cascade of cellular interactions leading to partial restoration of cell-mediated immune responses. These interferences with the defective helper/suppressor regulatory pathways may have important therapeutic implications.

IMMUNOLOGIC DYSFUNCTION AMONG PBB‐EXPOSED MICHIGAN DAIRY FARMERS*

In 1973 inadvertent contamination occurred in a special farm feed supplement for lactating cows. Polybrominated biphenyls (PBBs) were used in place of magnesium oxide resulting in serious harm to farm animals, including cattle, chickens, geese, ducks. Farm families, accustomed to eating their own products, were most heavily exposed. To further study the impact of PBBs, 45 adult Michigan farm residents who were originally examined in a clinical field survey were further studied with respect to their immunologic status. For comparison, 46 dairy farm residents in Wisconsin, who had not eaten PBB-contaminated food, were examined, as were 79 healthy subjects in New York City. Abnormalities in the Michigan group included significant decrease in absolute numbers and percentages of T and B-lymphocytes and increased number of lymphocytes with no detectable surface markers (null cells). Significant reduction of in vitro immune function was noted in 35--40% of the Michigan farm residents who had eaten food containing PBB. Despite the absence of any apparent numerical reduction, both T and B lymphocyte subpopulations of peripheral blood lymphocytes showed evidence of functional defect. Ten of the 45 Michigan farmers studied showed impaired PHA-induced blastogeneic response, due to the decreased number and percent of T-cells in the PBLs. The decreased immune function detected among the PBB-exposed farm residents tended to affect families as a unit and was independent of exposed individuals age or sex, speaking against the possibility of genetic predisposition.

Impaired immune function and identification of polybrominated biphenyls (PBB) in blood compartments of exposed Michigan dairy farmers and chemical workers.

In 1973 PBBs were accidentally mixed into animal feed, resulting in marked toxic effects. Meat and dairy products were widely consumed in Michigan. To determine the impact of PBBs, 55 exposed Michigan farm residents, 11 Michigan chemical workers and 46 non-exposed Wisconsin farmers were examined. Abnormalities included decreased number of T-lymphocytes with concomitant increase of lymphocytes with no detectable surface markers, null cells, and altered lymphocyte function. Data obtained from skin testing using standard recall antigens, showed no consistent correlation between the delayed cutaneous hypersensitivity response and the impaired lymphocyte function. PBB (hexa) in separated white blood cells and red cells was positively identified and quantified by gas chromatography-mass spectrometry. PBB and immunological abnormalities were not detected in non-exposed Wisconsin dairy farm residents.

Specific immunotherapy with neuraminidase-modified leukemic cells: experimental and clinical trials.

The data presented establish the therapeutic effectiveness of immunotherapy with neuraminidase-treated allogeneic myeloblasts in combination with sustaining chemotherapy in patients with acute myelocytic leukemia. The in vivo and in vitro immunologic tests indicate normal immunocompetence in patients receiving immunotherapy versus control patients treated with chemotherapy alone. These findings correlate well with the improved duration of remission as the direct result of the immunotherapy.

3-m-Bromoacetylamino benzoic acid ethyl ester : A new cancericidal agent that activates the apoptotic pathway through caspase-9

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.