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There is a disease stage-dependent loss of CD28 expression on T cells in HIV-infected children. In this study, T cell recovery, in particular CD28 expression on T cells, was analyzed after initiation of highly active antiretroviral therapy in a group of eight mostly treatment-naive HIV-infected children. Plasma HIV-RNA levels were recorded, and numbers of CD4, CD8, CD4+CD28+, and CD8+CD28+ cells were determined by two-color flow cytometry. Values after 12 mo of therapy were compared with age-matched, seronegative control subjects. CD4 recovery to subnormal values was observed in all children. CD8+CD28+ cells recovered and were within the normal range after 12 mo of therapy (patients, 703 +/- 250 cells/microL; controls, 789 +/- 269 cells/microL), whereas CD8+CD28- cells (546 +/- 269 cells/microL) remained significantly expanded compared with age-matched controls (140 +/- 35 cells/microL). Expansions of CD8+CD28- cells persisted even in cases with long-term suppression of viral replication. Highly active antiretroviral therapy in HIV-infected children induces substantial but incomplete T cell recovery.

Increased apoptosis of lymphocytes represents a key event of immune destruction in HIV infection. In this study it was investigated at which stage of the disease and in which T lymphocyte subpopulation (CD4+ or CD8+) protection against apoptosis may be lost as measured by decreased CD28 expression. In 26 HIV-infected and 20 healthy children, as well as 10 infants exposed to HIV, expression of CD28 and the apoptosis-related marker CD95 was studied by fluorescence-activated cell sorting analysis. According to established Centers for Disease Control and Prevention definitions, children were divided into three immunologic categories. In the CD8 population, patients in category 1 already showed a markedly decreased mean CD28 (36.2%+/-16.1 SD) and increased CD95 expression (48.8+/-24.1%), compared with the age-matched control group (67.7+/-14.4%, 15.8+/-8.9%). In the CD4 population, mean CD28 and CD95 expression was not altered in category 1 patients. Of the exposed children, the child with the lowest CD28 expression on CD8 cells was determined later to be infected with HIV. Significant immunophenotypical alterations are observed in early stage pediatric HIV infection, which may indicate an early loss of protection against apoptosis in the CD8+ T cell population.

Because the antiprotease defense in cystic fibrosis (CF) airways is overwhelmed by neutrophil elastase (NE), substitution of antiproteases such as secretory leukoprotease inhibitor (SLPI) seems to be a reasonable therapeutic approach. Knowing, however, that native antiproteases may be liable to rapid inactivation by the locally abundant oxidants, we comparatively investigated the interactions of CF sputum with recombinant native SLPI (rSLPI) and its partially oxidation-resistant variant (rSLPI-242), respectively, to estimate their therapeutic potentials. NE activity in supernatants from diluted CF sputum samples was dose-dependently inhibited by both rSLPI and rSLPI-242, with comparable potency. Addition of the oxidant N-chlorosuccinimide resulted in significant superiority of rSLPI-242 over rSLPI. When fresh neutrophil-rich CF sputum was incubated with rSLPI and rSLPI-242, respectively, rSLPI-242 inhibited sputum NE activity significantly more potently than did rSLPI; addition of the antioxidant superoxide dismutase significantly improved the effect of rSLPI. Furthermore, secretion of radiolabeled macromolecules from porcine tracheal glands induced by purified NE or by CF sputum was inhibited dose-dependently by rSLPI and even better by rSLPI-242. We conclude that both rSLPI and rSLPI-242 effectively inhibit NE activity and NE-induced gland hypersecretion in vitro. In vivo effects of CF remain to be analyzed; an advantage of the partially oxidation-resistant rSLPI-242 can be expected.

Sera from obligate heterozygotes for deficiency of the C8 beta subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8 alpha-gamma and C8 beta subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitometry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8 alpha-gamma (median 99 AU) and from 45 to 140 AU for C8 beta (median 101), whereas the C8 alpha-gamma/C8 beta-ratio varied from 0.7 to 1.4. Sera from C8 beta-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8 beta (20 to 90 AU, median 55 AU) and for the higher C8 alpha-gamma/C8 beta-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8 beta-bound C8 alpha-gamma. The migration of free and C8 beta-bound C8 alpha-gamma subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8 alpha-gamma using this system revealed about 5-14% free C8 alpha-gamma in sera with normal C8 and higher levels, from 33-71%, in the C8 beta D heterozygous sera. Functional analysis confirmed the substantial increase of free C8 alpha-gamma in the heterozygous group. We conclude that the C8 in C8 beta D heterozygous sera is characterized by increased amounts of free C8 alpha-gamma due to reduced concentrations of the C8 beta subunit. This finding may help to identify individuals heterozygous for C8 beta deficiency.