Quantification of human immunodeficiency virus type 1 p24 antigen and antibody rivals human immunodeficiency virus type 1 RNA and CD4+ enumeration for prognosis. National Institute of Child Health and Human Development Intravenous Immunoglobulin Clinical Trial Study Group.

BACKGROUND: The sensitivity, specificity and positive predictive value of baseline serum concentrations of HIV-1 immune complex-dissociated (ICD) p24 antigen for predicting disease progression and mortality were assessed and compared with results obtained for HIV-1 ICD p24 antigen with HIV-1 p24 antibody and for HIV-1 RNA with CD4+ lymphocyte percent. METHODS: Data from HIV-infected children enrolled in a North American clinical trial (National Institute of Child Health and Human Development Intravenous Immunoglobulin Clinical Trial) were analyzed. Disease progression was defined as growth failure, CD4+ lymphocyte percent decline to <15% after study entry or development of an AIDS-defining opportunistic infection. RESULTS: Baseline samples were available for ICD p24 antigen testing (median concentration, 319 pg/ml; range, <50 to 15,640) in 240 children. The combination of detectable ICD p24 antigen and low p24 antibody was more sensitive but less specific than the combination of high HIV-1 RNA and low CD4+ lymphocyte percent in predicting disease progression and mortality. Using receiver operating characteristic curves, the specificity of ICD p24 antigen with p24 antibody for classifying children's disease progression or mortality was as great as, or greater than, HIV-1 RNA with CD4+ lymphocyte percent at points on the curve corresponding to higher sensitivity. CONCLUSIONS: The use of ICD p24 antigen with p24 antibody to identify children at high risk of disease progression or mortality could be a viable alternative to the more expensive and technically difficult HIV-1 RNA and CD4+ lymphocyte assays in resource-poor settings, including developing countries where the majority of children with HIV-1 infection reside.

Reduction in T cell apoptosis in patients with HIV disease following antiretroviral therapy.

Patients with HIV infection manifest increased T lymphocyte apoptosis. This study investigated the influence of antiretroviral therapy (ART) upon lymphocyte apoptosis in 23 HIV-infected adults naive to protease inhibitors. Patients were enrolled in a treatment trial consisting of Nelfinavir (NFV), d4T, or NFV + d4T for 24 weeks, followed by triple therapy (NFV + reverse transcriptase inhibitors) for an additional 24 weeks. Spontaneous T cell apoptosis in cultured PBMC decreased by 23.67 +/- 18.2% (P < 0.006) at 48 weeks and plasma HIV RNA decreased by 1.79 +/- 0.59 log(10) RNA copies/ml (P < 0.001). The absolute decrease and slope of T cell apoptosis correlated with plasma virus load and with activated CD8 T cells and was inversely correlated with CD4 T cells. We conclude that reduction in chronic antigenic stimulation and the absence of cellular signals elicited by viral products contribute to the rescue of T lymphocytes from apoptosis, which facilitates immunologic recovery in ART-treated patients.

Plasma virus load evaluation in relation to disease progression in HIV-infected children.

The objective of this study was to investigate the relationship of plasma HIV RNA load with survival and disease progression in HIV-infected children and to determine its correlation with cellular HIV DNA. Virus load (VL, HIV RNA copies/ml) was determined retrospectively by nucleic acid sequence-based amplification (NASBA) assay in 144 stored plasma samples between birth and 48 months in 50 children of whom 40 are alive (age range, 2-13 years). On the basis of clinical and immunologic status children were classified as rapid progressors (RPs), or nonrapid progressors (NRPs). Proviral HIV DNA quantitated by QC-PCR (quantitative competitive polymerase chain reaction) in 24 children was compared with plasma HIV RNA. At age <3 months, plasma VL <750,000 copies/ml was associated with significantly higher survival to age >2 years (p < or =0.01) compared with a VL of > or =750,000 copies/ml. Increasing mortality was observed with increasing plasma HIV RNA levels at ages 3-24 months and baseline VL of infants who died before age 24 months was significantly higher (p = 0.004) than baseline VL of those who survived beyond 24 months. Although baseline VL in infants classified as RPs was higher than that of NRPs, the difference was not statistically significant. Among surviving children 2-13 years of age, the baseline VL obtained at <24 months of age was not predictive of disease severity. Although no significant correlation was noted between plasma HIV RNA and proviral DNA, the concurrence of positive and negative results was >80%. We conclude that high plasma HIV RNA in infancy is associated with increased mortality.

Distribution of CCR5delta32 in human immunodeficiency virus-infected children and its relationship to disease course.

Homozygosity for a 32-bp deletion in the CCR5 gene (CCR5delta32) has been shown to confer resistance to infection with the macrophage-tropic strain of human immunodeficiency virus (HIV) type 1. We examined the distribution of CCR5delta32 in 47 children (age range, 1.5 to 19 years), of whom 43 were infected with HIV, by the perinatal route (n = 41) or by the intravenous route (n = 2). The infected patients were classified as rapid progressors (RP) (n = 7) (CDC category C3 or death by 2 years of age), non-rapid progressors (NRP) (n = 17) (survival for > or =8 years after infection), or intermediate (n = 19). CCR5delta32 heterozygosity was found in two HIV-infected children, both NRP. None of the subjects were homozygous for CCR5delta32, and the remaining children had no evidence of CCR5delta32. The presence of CCR5delta32 heterozygosity in 4.8% of this, predominantly non-Caucasian population is consistent with the published distribution of the mutation. The finding that CCR5delta32 was present only in NRP and not in any RP is in agreement with previous reports suggesting that heterozygosity for CCR5delta32 may confer limited protection from disease progression.

Evaluation of pharmacokinetics, safety, tolerance, and activity of combination of zalcitabine and zidovudine in stable, zidovudine-treated pediatric patients with human immunodeficiency virus infection. AIDS Clinical Trials Group Protocol 190 Team.

A double-blind phase II trial compared zalcitabine (0.03 mg/kg/day) in combination with zidovudine (720 mg/m2/day) and zidovudine monotherapy in 250 clinically stable, previously zidovudine-treated, human immunodeficiency virus-infected children. The combination was well-tolerated except for an increased incidence of neutropenia (14%) compared with that in children receiving monotherapy (5%). No differences were noted for time to first AIDS-defining illness or death, neuropsychologic status, or weight Z scores. In patients in the combination arm, the CD4 cell count decline was slower (13% per year) than in patients receiving monotherapy (25% per year) (P = .03), and quantitative peripheral blood mononuclear cell virus load remained lower at all time points (P = .08). Deaths were fewer in patients receiving combination therapy (4) compared with those in patients receiving monotherapy (10) (P = .083). Thus, administration of zidovudine with zalcitabine to children with prior zidovudine treatment did not result in a significant increase in toxicity compared with that resulting from zidovudine monotherapy and demonstrated improvement in immunologic and virologic surrogate markers.

Outcome and survival in HIV-infected infants with Pneumocystis carinii pneumonia and respiratory failure.

A retrospective chart review (January 1987-December 1994) of cases of histologically proven Pneumocystis carinii pneumonia (PCP) in 9 infants (ages 1.1-7 months) who had perinatally acquired human immunodeficiency-1 virus (HIV) infection was performed. None of the children was suspected of having HIV or had received PCP prophylaxis. Respiratory failure requiring mechanical ventilation developed in all 9 children. Comparison of survivors (5) with nonsurvivors (4) showed no significant differences in the age of onset, weight for length, hemoglobin level, total protein/albumin, lactic dehydrogenase (LDH), liver function tests, lymphocyte numbers and functions, time on mechanical ventilation, treatment received (including the use of steroids), and other complications occurring during the acute phase of pneumonia. The survivors had significantly higher platelet counts than nonsurvivors (mean 516 K versus 237 K, p = 0.02), a trend toward lower arterial-alveolar (A-a) gradient (mean 415 versus 218, p = 0.07), and earlier use of steroids after the onset of illness (2.5 versus 1 day, p = 0.06). Four of 5 children treated after December 1989 survived compared to 1 of 4 prior to that. Four survivors followed for a median length of 29 months (range 28-32 months) had stable physical and neurocognitive development, improvement in CD4+ T cell counts [mean 27% (range 23-36%), absolute count-mean 1631 (range 1427-1631)] and immunologic functions, and decrease in p24 Ag in 3 of 4. The cellular proviral load measured by DNA quantitative polymerase chain reaction (QC-PCR) decreased (40 K to 17.3 K copies) in one of two patients studied at two time points. PCP continues to be a serious complication of HIV infection in infancy and aggressive preventive approaches seem warranted. In our institution no single factor was responsible for improved survival following PCP after 1989. Four of 5 survivors continued to do well 28-32 months after the acute episode.

Repeatedly positive human immunodeficiency virus type 1 DNA polymerase chain reaction in human immunodeficiency virus-exposed seroreverting infants.

Three human immunodeficiency virus type 1 (HIV-1)-exposed children who had repeatedly positive DNA polymerase chain reaction (PCR) tests for HIV in > or = 5 samples before seroreversion to HIV-negative status are reported. The children belong to a cohort of 210 infants who were born to HIV-infected mothers and were tested at intervals of 1 to 3 months by HIV viral culture, PCR, and p24 antigen; only the PCR was positive in > or = 5 samples in the children reported here. Their clinical features were indistinguishable from other seroreverters. All three children had a transient drop in CD4:CD8 ratio to < 1.0. The transiently positive DNA PCR in HIV-exposed infants may indicate either that HIV infection was eliminated by a strong host immune response or that infection was caused by an attenuated/defective strain of virus.

Detection of HIV-specific antibodies in infancy by isoelectric focusing and affinity immunoblotting.

Asymptomatic congenital HIV infection cannot be diagnosed in infants less than 15 mo by routine serologic techniques because of the presence of passively acquired maternal antibody in the infants' circulation. Possibly, infants who synthesize antibody to highly conserved HIV proteins may be recognized by the detection in serum of clonally distinct IgG antibodies to HIV. To test this hypothesis, isoelectric focusing in thin-layer agarose gels was combined with affinity immunoblotting to antigen-coated nitrocellulose membranes. In all 10 cases examined, the presence or absence of clonotypically distinct bands of IgG antibodies was concordant with infectious status. Thus, this technique may provide an accurate serologic approach to the diagnosis of congenital HIV infection.

Neoplastic complications of HTLV-III infection. Lymphomas and solid tumors.

Neoplastic disease arose in 29 of 200 patients infected with human T lymphotropic virus type III (HTLV-III) seen at a suburban hospital. Seventeen patients had Kaposi's sarcoma, one of whom also had colon carcinoma. Nine patients had lymphoproliferative disorders (seven lymphomas, one T suppressor cell chronic lymphocytic leukemia, and one multiple myeloma), including three with concomitant Kaposi's sarcoma and one with colon cancer. One other patient had colon cancer, one had a seminoma, and one had pancreatic cancer. Kaposi's sarcoma as a complication of AIDS occurred mainly in homosexuals (17 of 42 homosexuals, one of 17 drug abusers, one of five heterosexually promiscuous patients, and one of six patients who had previously received transfusions). The high-grade lymphomas did not show a predilection for any particular AIDS risk group. Three of four solid tumors arose in elderly AIDS patients. Twenty-five of 75 patients with CDC-defined AIDS had a neoplastic disorder (26 are still alive and may yet demonstrate malignancy). Few other diseases of man have been associated with as high an incidence of neoplastic transformation as occurs with HTLV-III infection.

Unusual features of scabies complicating human T-lymphotropic virus type III infection.

Four cases of "exaggerated scabies" are described in patients who were immunodeficient secondary to human T-lymphotropic virus type III (HTLV-III) infection. As in classical scabies, these patients had a pruritic dermatitis but lacked the usual distribution of the eruption. The rash was initially misdiagnosed in all four patients. Scabies should be considered in pruritic dermatitis in patients at risk for HTLV-III infection.

A classification of HTLV-III infection based on 75 cases seen in a suburban community.

Since 1981, 75 patients have been seen at our hospital with human T-cell lymphotropic virus type III (HTLV-III) infection. We have classified their clinical presentation into Groups 0 to 6. Groups 0 to 3 all have antibody to the Mr 41,000 protein of HTLV-III. Group 0 has no evident disease (9 patients), Group 1 has lymphadenopathy with or without exaggerated infection (16 patients), Group 2 has persistent lymphadenopathy with chronic hepatitis B surface antigenemia or profound hypergammaglobulinemia (7 patients), Group 3 has oral candidiasis with or without lymphadenopathy (7 patients). In Group 4 are acquired immunodeficiency syndrome (AIDS) adults or children (32 patients). Group 5 is a special classification for immunocompromised patients. Group 6 patients have lymphomas and Mr 41,000 protein antibody. Four children were classified separately. Three patients in Group 3 developed Group 4 disorders (AIDS). Four patients in Group 4 developed Group 6 disorders. HTLV-III infection spread in families (8 of 36), all from infected mothers to children. In 17 sexual partners, 6 were found to be infected. Five of 6 infected partners were homosexuals. We saw an inordinate number of transfusional AIDS (4 of 29) and 1 of 46 other disorders. Two infants also presented with severe intracranial defects, one with microcephaly and one with cranial calcifications and lucency. HTLV-III is spreading with alarming speed.

Defective B-lymphocyte function in homosexual men in relation to the acquired immunodeficiency syndrome.

Patients with the acquired immunodeficiency syndrome manifest a wide spectrum of immunologic abnormalities. Polyclonal and antigen-specific differentiation of B lymphocytes into immunoglobulin- and antibody-secreting cells was studied in vitro in three groups of homosexual volunteers: asymptomatic men; men who were symptomatic but lacked clinical criteria for the acquired immunodeficiency syndrome; and those having the syndrome. Although mean responses of all three groups were significantly lower than those of healthy heterosexual male controls, those of the asymptomatic group were least affected. Abnormalities in the symptomatic group were equal to or greater than those of patients with the syndrome, but responses of the latter group were the least augmentable by in vitro manipulations. Severe impairment of B-cell function appeared to favor clinical deterioration. Antibody replenishment might be of value as adjunctive therapy in persons with the acquired immunodeficiency syndrome or as prophylaxis in certain at-risk persons.

Immunoregulatory properties of childhood leukemias.

Investigation of in vitro humoral immune responses and immunoregulatory properties of leukemic cell was carried out in 17 children with acute leukemia prior to therapy. Leukemias were of the non-T, non-B-cell type in 13 patients and of T-cell origin in four. Bone marrow and peripheral blood cells consisted of 24-96% lymphoblasts and were generally deficient in surface Ig-positive cells. Induction of Ig secreting cells in response to pokeweed mitogen was markedly decreased in marrow and peripheral mononuclear cell cultures of leukemic patients. Co-culture of leukemic cells with normal lymphocytes led to marked deviations from the expected Ig secreting-cell response of the cell mixtures. The predominant effect was enhancement, as was the case with eight non-T, non-B-cell and one T-cell leukemia samples. Suppression of the Ig secreting-cell response was observed in only three instances, two with non-T, non-B-cell and one with T-cell leukemia samples. These findings implicate non-T, non-B as well as more differentiated leukemic cells in having the potential for modifying Ig production by B cells.

Polyclonal and antigen-specific B-cell responses in patients with common variable immunodeficiency.

Antigen-specific antibody responses were investigated in 32 hypogammaglobulinemic patients with common variable immunodeficiency following in vitro sensitization of their peripheral blood lymphocyte cultures with sheep red blood-cell determinants. Anti-sheep red blood-cell antibody-secreting cells were quantitated in a hemolytic plaque assay. Amplification of T-cell help was achieved with the use of the T-cell mitogen concanavalin A or allogeneic irradiated T cells. Four patients groups, A through D, were identified. Group A was comprised of 10 patients whose cultured lymphocyte readily developed into antibody secreting cells. Cultures of 9 patients (Group B) responded suboptimally, but were enhanced following mitogen activation of autologous or exogenous T cells, and those of 7 patients (Group C) responded only when help was amplified. In 7 patients (Group D), no responses were elicited. On the simultaneous assessment of pokeweed mitogen-driven polyclonal generation of immunoglobulin-secreting cells, only 10 responders, all from groups A and B, were identified. Our observations indicate that the majority of patients with common variable immunodeficiency possesses B cells capable of producing antibody in vitro. The ability of some patients' B cells to respond only in the antigen-specific assay while failing to do so in pokeweed mitogen-stimulated cultures suggests that these two reactions are not identical in their activation pathways.

Abnormal humoral immune responses in peripheral blood lymphocyte cultures of bone marrow transplant recipients.

The present study was aimed at investigating recovery of humoral immunity in vitro after bone marrow transplantation in patients with acute leukemia and severe aplastic anemia. Hemolytic plaque assays were utilized to quantitate pokeweed mitogen-stimulated polyclonal immunoglobulin production and sheep erythrocyte antigen-specific antibody responses in cultures of peripheral blood mononuclear cells of 39 patients beginning at 1 month, for variable periods up to a maximum of 4 years after marrow transplantation. Three phases were identified: an early period of primary B cell dysfunction with concomitant immunoregulatory T cell abnormalities--i.e., decreased helper and increased suppressor activities; an intermediate phase in which B cell dysfunction could be attributed in large measure to immunoregulatory T cell abnormalities; and a late phase of normal B and T lymphocyte functions. Patients with graft-versus-host disease differed from those without it in that they often did not manifest increased T cell suppressor activity in the early period, and they were noted to have prolonged and profound B and T cell abnormalities in the chronic phase of their disease. In selected patients, simultaneous assessment of ratios of Leu-2 to Leu-3 antigens on T cells by monoclonal antibodies and of immunoregulatory T cell functions revealed a correlation between the two only late in the post-transplant period. These studies provide an insight into the ontogeny of B cell function in the post-transplant period and indicate that in certain situations phenotypic alterations in T cell subsets cannot reliably be used to predict abnormalities in their function in recipients of marrow transplantation.

The role of an adherent cell in the production of monocyte chemotactic factor.

The functional role of an adherent cell in assisting a variety of in vitro immune responses is well established. An assay for human monocyte chemotaxis in vitro was utilized as a means of investigating the role of adherent cells in the production of the monocyte chemotactic factor that is produced by peripheral blood lymphocytes upon stimulation with Concanavalin A. Depletion of a population of adherent cells by passage of peripheral blood mononuclear cells through a Sephadex G-10 column rendered the latter incapable of producing monocyte chemotactic factor. The requirement for adherent cells in the production of a "lymphokine" is in agreement with many previous works of a similar nature performed in other experimental systems.

Decreased in vitro humoral immune responses in aged humans.

Induction of antigen-specific and non-specific (polyclonal) humoral immune responses in vitro was investigated in peripheral blood mononuclear cells of aged (65-85 yr) and young (20-30 yr) volunteers. In vitro immunization of lymphocytes with antigen (sheep erythrocytes) was performed in a recently described microculture system, and anti-sheep erythrocyte plaque forming cells were quantitated in a direct hemolytic plaque assay. Immunoglobulin secreting cells, induced polyclonally with pokeweed mitogen, were quantitated in a reverse hemolytic plaque assay. Significant depressions of antigen-specific as well as polyclonal responses were noted in relation to advancing age. Antigen-specific responses were more frequently depressed than polyclonal responses. T cell mitogen concanavalin A (Con A) was used to amplify functions of autologous immunoregulatory T cells. Addition of 10 microgram/ml Con A to lymphocytes of young donors at culture initiation resulted in activation of suppressor cells and abrogated antigen-specific responses. Delayed addition of Con A, on the other hand, enhanced responses, presumably because of activation of helper T cells. Similar manipulations of lymphocyte cultures from aged donors showed failure of Con A to suppress antigen-specific responses in approximately half of the responders. In many nonresponders, responses within normal range were elicited by the delayed addition of Con A to their lymphocyte cultures. Deviations beyond the range of expected responses were noted in 32.5% of the co-cultures between pokeweed mitogen stimulated young and aged cells. Our findings suggest that age-related deficiencies of B cell function are frequently associated with dysfunction of immunoregulatory T cells and are only occasionally due to intrinsic defects of B cells.

The role of mitogens and antigens in the generation of antibody-producing human B lymphocytes.

Satisfactory experimental systems with which to study the antigen specific humoral immune response of human peripheral blood lymphocytes have not been available until recently. A commonly used method for the study of antibody production by human lymphocytes is that developed by Fauci and Pratt. This system is considered to be antigen nonspecific since the antigen against which the determined antibody is directed is not added to the cultures. We show here that the assumption of the Fauci-Pratt system being antigen nonspecific is not justified. An essential ingredient of this culture system is human serum that has been exhaustively absorbed with antigen (sheep red blood cells). This absorption procedure causes shedding of highly immunogenic antigenic fragments whose immunogenic activity we demonstrated using a recently developed antigen-dependent culture system. In the latter system, we have shown that the control of suppressor cells is a critical factor for the successful induction of antibody responses, particularly in view of the fact that lymphocyte mitogens must be added to cultured human lymphocytes to support their responsiveness. Appropriate timing of mitogen addition to the cultures was found to be an effective means of preferentially stimulating helper or suppressor activity. Pokeweed mitogen, a mitogen known to act on B and on T lymphocytes, Stimulates B cells responses readily but abrogates them prematurely by the simultaneous activation of suppressor cells. When pokeweed mitogen is added to an ongoing response with a delay of 48 hr, it enhances antibody responses markedly, presumably by providing additional help to B cells at a time when they have lost susceptibility to suppressor-cell effects.