Effects of different HIV strains on thymic epithelial cultures.

The thymus is the major site for T cell development; interactions of developing thymocytes with thymic epithelial cells are critical for normal thymopoiesis. We set out to determine whether thymic epithelial cells can be infected by HIV strains that cause different patterns of disease progression in infected infants. Thymic epithelial cell monolayers were prepared from normal thymus of infants, removed at the time of cardiac surgery. We infected the thymic epithelial cell monolayers with different strains of HIV, including laboratory strains and clinical strains from 3 of our pediatric HIV-infected patients with different patterns of disease progression. We found that different strains of HIV have different ability to infect thymic epithelial cells; the ability to infect thymic epithelial cells may not be directly related to CCR5/CXCR4 usage. Different HIV strains thus appear to employ different mechanisms by which they affect thymic components.

Use of human CD3 monoclonal antibody for accurate CD4+ and CD8+ lymphocyte determinations in macaques: phenotypic characterization of the CD3- CD8+ cell subset.

Macaque monkeys are frequently used in models for studies of infectious diseases, immunity, transplantation and vaccine development. Such use is largely due to the conservation of functionally important cell surface molecules and the phylogenetic proximity of their immune systems to that of humans. Some monoclonal antibodies (mAb) raised against human leukocyte antigens can be utilized in the monkey. Until recently, many primate centers have utilized the CD2 monoclonal antibody to enumerate T lymphocytes. We have evaluated the anti-human CD3 mAb in macaques and sooty mangabeys. Using this monoclonal antibody, pigtailed macaques were found to have a much higher proportion of CD2+ CD3- CD8+ cells as compared with rhesus macaques and sooty mangabeys. Such cells comprised approximately one-half of all CD8+ cells in the pigtailed macaque, but only one-quarter of CD8+ cells in the rhesus, and one-fifth in the sooty mangabey. Use of the CD2 monoclonal antibody as the T-cell marker resulted in underestimating CD4/CD8 ratios compared with using the CD3 mAb in pigtailed macaques. Phenotypic characterization of this subset of CD3- CD8+ cells indicated that they are CD16+, CD45RA+, CD11b+, CD69+ and CD28-. This would indicate that these cells represent an activated natural killer cell subset.

Prolonged dominance of clonally restricted CD4(+) T cells in macaques infected with simian immunodeficiency viruses.

The repertoire of functional CD4(+) T lymphocytes in human immunodeficiency virus type 1-infected individuals remains poorly understood. To explore this issue, we have examined the clonality of CD4(+) T cells in simian immunodeficiency virus (SIV)-infected macaques by assessing T-cell receptor complementarity-determining region 3 (CDR3) profiles and sequences. A dominance of CD4(+) T cells expressing particular CDR3 sequences was identified within certain Vbeta-expressing peripheral blood lymphocyte subpopulations in the infected monkeys. Studies were then done to explore whether these dominant CD4(+) T cells represented expanded antigen-specific cell subpopulations or residual cells remaining in the course of virus-induced CD4(+) T-cell depletion. Sequence analysis revealed that these selected CDR3-bearing CD4(+) T-cell clones emerged soon after infection and dominated the CD4(+) T-cell repertoire for up to 14 months. Moreover, inoculation of chronically infected macaques with autologous SIV-infected cell lines to transiently increase plasma viral loads in the monkeys resulted in the dominance of these selected CDR3-bearing CD4(+) T cells. Both the temporal association of the detection of these clonal cell populations with infection and the dominance of these cell populations following superinfection with SIV suggest that these cells may be SIV specific. Finally, the inoculation of staphylococcal enterotoxin B superantigen into SIV-infected macaques uncovered a polyclonal background underlying the few dominant CDR3-bearing CD4(+) T cells, demonstrating that expandable polyclonal CD4(+) T-cell subpopulations persist in these animals. These results support the notions that a chronic AIDS virus infection can induce clonal expansion, in addition to depletion of CD4(+) T cells, and that some of these clones may be SIV specific.

Correlation of virus load and soluble L-selectin, a marker of immune activation, in pediatric HIV-1 infection.

OBJECTIVE: HIV infections in children are characterized by high viral load and, in some perinatally infected newborns, delayed appearance of viral markers. Both phenomena may be related to different levels of immune activation affecting viral replication. This study was designed to investigate the relationship between immune activation and viral replication in pediatric HIV infection, and the role of pre-existent immune activation in facilitating HIV transmission to the fetus/newborn. DESIGN: Plasma levels of soluble L-selectin (s-LS), an immune activation marker, were determined in 100 infants with perinatally transmitted HIV infection, compared with 106 age-matched HIV-exposed uninfected controls. Included in the analysis were samples from 31 HIV-infected (10 PCR+ and 21 PCR-) and 35 uninfected newborns aged < 2 days. METHODS: To determine s-LS levels, a solid phase ELISA was performed on plasma samples of patients and controls. RESULTS: s-LS levels in uninfected children were higher than those in normal adults. HIV-infected patients had more rapidly increasing values in the first 6 months of life compared with uninfected infants. Plasma s-LS levels correlated with HIV viral loads (r, 0.50). Among newborns in the first 2 days of life, s-LS levels were lowest in those with negative PCR tests, compared with PCR-positive or uninfected infants. CONCLUSIONS: These results suggest that higher immune activation in children contributes to higher viral loads, and that the level of pre-existent immune activation may have a role in determining which infants have detectable virus in peripheral blood at birth.

The disruption of macaque CD4+ T-cell repertoires during the early simian immunodeficiency virus infection.

T-cell receptor (TCR) complementarily determining region 3 (CDR3) spetratyping analysis was employed to assess the ability of an AIDS virus to disrupt CD4 + T-cell repertoires during the primary infection. Rhesus and pig-tailed macaques infected with simian immunodeficiency virus (SIV)mac 251 and SIVsmmFGb, respectively, were evaluated. Following SIV infection, the macaques exhibited an apparent decline of CD4 + peripheral blood lymphocyte (PBL) counts, which was associated with a change in CDR3 profiles from multiple-length distribution to one- or two-length dominance in the selected TCR Vbeta-expressing CD4 + PBL subpopulations. Molecular analysis of the perturbed cell subpopulations suggested that the CD4 + T cells bearing the dominant CDR3 length were clonally expanded. These results indicate that SIV infection can induce a disruption of macaque CD4 + T-cell repertoires during the primary infection. The finding in this study, therefore, suggests that the virus-induced clonal dominance can contribute to the disruption of CD4 + T-cell repertoires.

Thymic dysfunction and time of infection predict mortality in human immunodeficiency virus-infected infants. CDC Perinatal AIDS Collaborative Transmission Study Group.

The effect of human immunodeficiency virus (HIV)-induced thymic dysfunction (TD) on mortality was studied in 265 infected infants in the CDC Perinatal AIDS Collaborative Transmission Study. TD was defined as both CD4 and CD8 T cell counts below the 5th percentile of joint distribution for uninfected infants within 6 months of life. The 40 HIV-infected infants with TD (15%) had a significantly greater mortality than did the 225 children without TD (44% vs. 9% within 2 years). Infants with TD infected in utero had higher mortality than did those infected intrapartum (70% vs. 37% within 2 years), while no significant difference was noted between infants without TD with either mode of transmission. The TD profile was independent of plasma virus load. Virus-induced TD by particular HIV strains and the time of transmission are likely to explain the variation in pathogenesis and patterns of disease progression and suggest the need for early aggressive therapies for HIV-infected infants with TD.

Enlargement of the thymus in a child with chronic granulomatous disease receiving interferon gamma therapy.

Both an enlarged thymus (with normal results on histologic examination) and an increase in the percentage of peripheral CD4+CD45RA+ (naive) T lymphocytes developed in a child with chronic granulomatous disease receiving long-term interferon gamma therapy. The thymic regrowth may be secondary to interferon gamma therapy or to overstimulation of his compromised immune system by recurrent infections. To our knowledge, an association between enlargement of the thymus and either chronic granulomatous disease or interferon gamma has not been previously reported.

Decreases in CD5+ B lymphocytes in children with the DiGeorge anomaly.

The authors evaluated the lymphocyte subsets in eight children with the DiGeorge anomaly, compared with 48 age-matched control infants. Of particular interest was the finding that the percentage and number of CD5+ B lymphocytes were decreased in seven of the eight cases. This observation may provide insight into thymic function and the interaction of the B and T cell systems in some forms of congenital and acquired immunodeficiencies.

Early progression of disease in HIV-infected infants with thymus dysfunction.

BACKGROUND: Infants with congenital thymic deficiency (the DiGeorge syndrome) have immunodeficiency and a characteristic pattern of low CD4+ and CD8+ T-lymphocyte counts and low CD5+ B-lymphocyte counts. Because the thymus is essential for the generation of CD4+ cells, we sought evidence of thymus dysfunction in infants infected perinatally with the human immunodeficiency virus (HIV). METHODS: We studied the immunophenotypes of 59 infants with maternally transmitted HIV, 5 infants with the DiGeorge syndrome, and 168 infants exposed to HIV but not infected. The criteria for a presumed thymic defect were reductions in both the CD4+ and CD8+ T-cell subgroups during the first six months of life that were confirmed in a subgroup of infants by low counts of CD4+CD45RA+ and CD4+CD45RO+ T cells and CD5+ B cells. RESULTS: Of the 59 HIV-infected infants, 17 had immunophenotypes similar to those of infants with the DiGeorge syndrome. The risks of the acquired immunodeficiency syndrome (AIDS) by the ages of 12 and 24 months were 75 percent and 92 percent in these 17 infants, as compared with 14 and 34 percent in the other 42 infants (P<0.001). Nine of the HIV-infected infants with the DiGeorge-like immunophenotype (53 percent) died within six months of the progression to AIDS, as compared with only three of the other infants (7 percent, P=0.006). CONCLUSIONS: In some infants infected perinatally with HIV, a pattern of lymphocyte depletion develops that resembles the pattern in congenital thymic deficiency. Since HIV disease progresses rapidly in such infants, they may be candidates for early antiviral therapy and attempts at immune reconstitution.

Subpopulations of T and B cells in perinatally HIV-infected and noninfected age-matched children compared with those in adults.

Peripheral blood mononuclear cells were quantified for the subsets of CD4, CD8, and CD19 lymphocytes by using CD45RA (2H4), CD29(4B4), CD57, CD5, CD10, Leu8, HLA-DR, and TCR gamma delta-1 monoclonal antibodies and dual color immunofluorescence. A comparative analysis of lymphocyte subpopulations was made among 52 HIV-infected and 50 age-matched control children and 30 HIV-seropositive and 27 negative control adults. A significant decrease in the CD4+CD45RA+ "naive" cells was much more marked in HIV-infected children than in HIV-infected adults. A significant percentage increase in the CD4+CD29+ "memory" cells was observed in HIV-infected children but not in infected adults; however, the absolute numbers were usually decreased in all age groups. The mean percentage and absolute numbers of CD4+CD7+ and CD4+Leu8+ cells were decreased in HIV-infected children, although usually not significantly. The CD3+TCR gamma delta-1+ did not show any change in the infected children tested. The mean percentage and absolute number of the CD8+HLA-DR+ cells increased significantly in HIV-infected persons of all ages. The CD8+CD57+ cells were increased in percentage and absolute number in HIV-infected children ages 1-4 and 4-8 years. In the adults, no change was noted in either the percentage or absolute number of CD19+CD5+ B cells, a finding similar to that noted in HIV-infected children above 1 year of age. Although adults showed a significant decrease in both percentage and numbers of CD5- B cells, an increase was noted in the 7- to 12-month-old HIV-infected children. The CD19+CD10+ cells showed a slight but significant decrease in the youngest age group and a significant increase in the older age groups of HIV-infected children. These findings indicate that several lymphocyte subpopulations are altered differentially during HIV infection in children of varying ages and in adults.

The development of the immune system--importance in the ascertainment of immunophenotypic changes in perinatal HIV infection.

Evidence is presented that the percentage and number of some subsets of T and B cells in normal children and adults vary greatly from those in fetal life and throughout the first few years after birth, and less so during adolescence and adulthood. Depending then on the age at which immunological studies are performed, as well as whether the HIV infection occurs in utero, at birth, or postnatally, values obtained by immunophenotypic analyses of differentiating or mature immunocytes will vary greatly. A concerted effort needs to be made to measure different developmental and activation immunophenotypic markers, from birth on, in premature and full-term infants of varying socioeconomic and ethnic background. Results from such studies should improve our ability to determine the timing of HIV infection, to obtain earlier guidelines for prophylaxis or treatment of the virus or of opportunistic infections, as well as to improve prognostic capabilities in perinatal HIV infection.

Analysis of synergism/antagonism between HIV-1 antibody-positive human sera and soluble CD4 in blocking HIV-1 binding and infectivity.

We tested human immunodeficiency virus type 1 (HIV-1) antibody-positive human sera and sCD4, alone and in combination, for synergistic, additive, or antagonistic effects on blocking of HIV binding and infectivity. Data were analyzed by an application of the median effect principle derived from the law of mass action. This allows the assessment of synergism/antagonism at any desired level of effect. Using three assays (whole virus binding to CD4 cells, neutralization of HIV infectivity, and binding of purified gp120 to solid-phase sCD4), we generally observed additive effects or slight synergism between antibody and sCD4 in inhibiting gp120-CD4 interaction. We used a fourth assay to measure the irreversible inactivation of HIV infectivity by sCD4, a property that can also be mediated by antibody but with considerably less potency than sCD4. The reduction in HIV infectivity mediated by mixtures of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. The relevant antiviral effects of sCD4 and anti-HIV sera may include reversible blockage of receptor binding, irreversible inactivation of HIV infectivity, and in the case of antibody, additional reactions that are independent of receptor binding. Although predictions concerning the in vivo situation are speculative, we find no evidence in vitro for antagonism between sCD4 and antibody with respect to the net effect of the two in blocking HIV binding and infectivity.

Structural features of CD4 required for binding to HIV.

A soluble form of the human CD4 glycoprotein (sCD4), the cellular receptor for human HIV, was treated with various physical, chemical, and enzymic regimens and tested over a range of concentrations for its capacity to inhibit the binding of HIV to CD4+ T cells. Reduction of disulfide bonds and alkylation in denaturing buffer (8 M urea) destroyed the inhibitory activity of sCD4, whereas reduction and alkylation in PBS had no effect. Derivatization or digestion of carbohydrate groups by periodate oxidation or by glycolytic enzyme digestion did not affect sCD4 inhibitory capacity. Digestion with trypsin or endoproteinase Glu-C destroyed activity. A limited digestion of sCD4 with endoproteinase Glu-C resulted in a mixture of fragments, however, and the mixture had inhibitory activity equivalent to that of intact sCD4. Within this mixture, a fragment of 23 kDa was identified that binds to HIV. Although sCD4 can be digested to yield fully active fragments, the requirement for intrachain disulfide bonding indicates that the minimum sized portion of CD4 that will retain full affinity for HIV will have to be formulated with a proper tertiary structure.

Studies on the antigenic specificity of Mycobacterium leprae. II. Immunological characterization.

Antigens from untreated and autoclaved Mycobacterium leprae obtained through chromatofocusing were tested for their ability to both induce as well as elicit skin reactivity in guinea pigs sensitized either with homologous and heterologous mycobacteria or with the fractions derived from autoclaved M. leprae. In former studies, of the several antigen-positive fractions, one showed specific activity and the remaining others cross-reactivity, as indicated by studies of hypersensitivity. The fraction exhibiting specificity contained only one antigen, whereas the other fractions contained more than one antigen. Because of the technique employed, all the fractions contained antigens that were proteinic in nature. In the latter studies, the antigens obtained from autoclaved M. leprae were shown to possess sensitizing capabilities, in addition to induction properties. Two fractions from untreated M. leprae had antigens that were heat-labile, whereas the remaining fractions contained heat-stable antigens.

Studies on the antigenic specificity of Mycobacterium leprae. III. Further studies on immunological characterization.

The ability of the various protein antigens of Mycobacterium leprae to induce as well as detect delayed type hypersensitivity has been confirmed by studies in mice. Additionally, one of the fractions obtained from untreated M. leprae has been shown to possess specificity to the organism through immuno-analysis, thus confirming previous observations on skin-reactivity in guinea pigs. SDS-PAG electrophoresis has shown that this fraction contains a single antigen. A suggestion has been made that this single protein could be a target antigen for early diagnosis of leprosy, specifically in contacts of leprosy patients. It could also assist in detecting a latent infection. Additional studies, using different parameters, should lead to further confirmation of its specificity. It has also been suggested that such M. leprae-specific protein antigens could play an important role in the immune response of leprosy patients. They could also have a significant impact as possible immuno-protective agents, either by themselves or in combination with other immuno-potentiating agents.

Studies on the antigenic specificity of Mycobacterium leprae. I. Isoelectric and chromatofocusing separation.

Cell sonicates of Mycobacterium leprae and other mycobacteria were subjected to isoelectric focusing and chromatofocusing to evaluate their protein antigens and to determine if the patterns were significantly different. Isoelectric focusing showed that the proteins of all mycobacteria focused within the pH range of 3.5 to 5.5, except those of M. leprae which extended beyond 5.5 to 6.5. These studies have indicated for the first time that the protein antigens of mycobacteria are acidic in nature. Comparison between the proteins of untreated and autoclaved M. leprae showed distinct differences between the two preparations, in respect of loss of some antigens in the autoclaved M. leprae sonicate. This indicates that the bands that were not visible in the autoclaved M. leprae were those of heat-labile proteins. It is possible, however, that the absent bands could have been of a low order of intensity and hence were not discernible. On the other hand, the proteins could have coagulated due to the heat treatment, thus causing confirmational changes or ionic interactions with membrane components, due to their acidic nature. It is possible that the proteins in the autoclaved M. leprae are the ones that possess immunogenic properties since the protective ability of heat-killed M. leprae has already been established. Chromatofocusing studies have confirmed the isoelectric focusing data in respect of the number of antigens and their respective protein content, besides permitting the availability of the various fractions for further biological characterization.