Early detection of antibodies against rDNA-produced HIV proteins with a flow cytometric assay.

There is evidence that some human immunodeficiency virus (HIV)-infected individuals have prolonged periods of seronegativity. A flow cytometric immunoreactive bead (IRB) assay is described for quantitative, simultaneous, and early detection of antibodies to HIV. Polystyrene beads of four diameters, each size coated with a different HIV recombinant DNA-produced protein (p24, p31, gp41, or gp120), bound anti-HIV antibodies detected with fluorescent antiglobulin. The IRB assay was performed on a panel of blood donor samples, many giving consistently false-positive enzyme immunoassay (EIA) and indeterminant Western blot (WB) results. The IRB assay proved as sensitive and more specific than currently licensed EIA and WB tests. Results on serial samples from eight HIV-infected individuals indicated that quantitation of anti-p24 by IRB assay may be useful in monitoring disease progression. Sequential pre- and post-EIA seroconversion sera from 35 HIV-infected homosexual men were tested by the IRB assay using IgM- and IgG-specific fluorescent probes. All 35 cases were IRB assay positive for at least one rDNA-p either before (17 of 35, 49%) or at the time of EIA positivity. Eleven cases (31%) initially had only IgM anti-HIV, primarily to gp41 (17%). In two individuals, the IgM response was detected at least 18 months before EIA seroconversion. The IRB assay is a widely applicable analytic procedure, potentially useful in pretransfusion anti-HIV screening of blood.

Concurrent enhancement of monocyte immunoregulatory properties and effector functions by recombinant interferon-gamma.

Interferon-gamma is a critical factor in the activation of several mononuclear phagocyte effector and immunoregulatory properties. However, it remains uncertain if IFN-gamma is capable of concurrent activation of both functions in the same cell population. Plastic adherent mononuclear cells (80-98% MN by cytochemical criteria) were cultivated in the absence or presence of recombinant interferon-gamma (rIFN-gamma, 0.1-100 U/ml) for 48 hr. MN surface DR antigen was assessed by flow cytometry (EPICS V) after staining with monoclonal antibodies OKIa1 or L243. Exposure to rIFN-gamma (100 U/ml) increased MN surface DR antigen (mean fluorescence intensity) by 80 +/- 20% (P less than 0.01) and 121 +/- 52% (P less than 0.001), respectively, compared to untreated cells. The increase in DR antigen was maximal at 100 U/ml, dependent on protein and RNA synthesis and blocked by agents that increase cAMP levels. IL-1 activity was determined in the mouse thymocyte assay; rIFN-gamma (100 U/ml) increased IL-1 activity in the supernatants of MN cultured in medium alone from 0.5 +/- 0.2 to 7.8 +/- 4.7 U/ml (P less than 0.05), and lipopolysaccharide-stimulated MN from 20.4 +/- 19.1 to 71.7 +/- 38.9 U/ml (P less than 0.05). Following rIFN-gamma exposure, MN stimulation of the AMLR was increased at 6 days (29,269 +/- 5224 vs 13,252 +/- 4938 cpm, P less than 0.01). Spontaneous cytotoxicity (SC) and antibody-dependent cell cytotoxicity (ADCC) were studied in a 51Cr release microculture assay using the human lymphoblastoid cell line CCRF-CEM as target. SC by MN increased linearly as a function of log[rIFN-gamma] for effector:target (E:T) ratios of 5:1 (r = 0.95, P less than 0.01) and 10:1 (r = 0.99, P less than 0.01). ADCC by MN increased following rIFN-gamma exposure (100 U/ml) at E:T ratios of 5:1 (22 +/- 13 to 31 +/- 4%, P less than 0.025) and 10:1 (31 +/- 4 to 38 +/- 4%, P less than 0.01). Thus, rIFN-gamma not only activates MN effector function, but has concurrent stimulatory effects on multiple MN properties critical to immunoregulation.

Identification of children requiring radiologic evaluation for urinary infection.

Sixty-nine children younger than 13 years of age with urinary tract infection were evaluated to identify risk factors for treatable urologic problems; i.e. those requiring surgery or prolonged antibiotic prophylaxis. All children had a renal ultrasound, intravenous pyelogram and voiding cystogram performed 4 to 6 weeks after the infection. Eleven children with treatable problems were identified, 10 with vesicoureteral reflux and 1 with a ureterocele. For identification of treatable problems the predictive value of a positive test was: (1) fever, 10 of 24 (41.7%); (2) abnormal D-deaminoarginine vasopressin renal concentrating ability, 8 of 24 (33.3%); (3) serum C-reactive protein greater than or equal to 1.0, 8 of 25 (32.0%); (4) Elevated urine N-acetylglucosaminidase, 5 of 16 (31.2%); (5) erythrocyte sedimentation rate greater than or equal to 25, 6 of 21 (28.6%); and (6) age less than 5 years, 10 of 43 (23.3%). Absence of fever denotes a low risk (less than 3%) of finding a treatable problem. Afebrile girls older than 5 years of age can have radiologic evaluation deferred until infection recurs. The presence of fever indicates a high risk of treatable urologic problems (41.7%) and warrants complete radiologic evaluation with the first urinary infection.

Capping of the surface OKT3 binding molecule prevents the T-cell proliferative response to antigens: evidence that this molecule conveys the activation signal.

The human T-lymphocyte receptor for antigen appears to have been localized to a cluster of associated surface glycoprotein molecules, among which is the OKT3 binding molecule. We have tested the hypothesis that selective removal of the OKT3 binding molecule eliminates the cellular immune response to antigens by clearing the surface of the molecule that conveys the activation signal. Enriched T cells were obtained from donors immune to purified protein derivative (PPD), streptokinase-streptodornase (SK-SD), or keyhole limpet hemocyanin (KLH). T-3 molecules were cleared from the cell surface by capping with OKT3 and F(ab')2 goat anti-mouse IgG. Regeneration of surface molecules was prevented by culturing the T-3 capped cells with OKT3 625 ng/ml. The capacity of capped T cells to proliferate in culture with antibody in response to antigens, alloantigens, and the mitogens, PHA and ionophore A23187, was compared to uncapped cells pretreated with media and to capped cells permitted to regenerate the OKT3 binding molecule. T-3 capped cells cultured in the presence of antibody failed to proliferate to antigens or alloantigens. However, T-3 capped cells cocultured with antibody also did not significantly proliferate to PHA, but did respond to A23187. In contrast, both media-treated T cells and cells which had regenerated the OKT3 binding molecule proliferated to mitogens, antigens, or alloantigens. The requirement for the OKT3 binding molecule was determined by utilizing T-1, T-4, and T-8 capped cells. T-1, T-4, or T-8 capped cells cultured in the presence of OKT1, OKT4, or OKT8 proliferated in response to antigens, alloantigens, and mitogens. These results demonstrate that in the absence of the OKT3 binding molecule, antigens, alloantigens, and PHA failed to induce a significant cellular proliferative response. In the absence of this molecule, PHA cannot bind to its carbohydrate moiety, and therefore cannot activate T cells to proliferate. These data support the concept that the molecule binding OKT3 conveys the transmembrane signal resulting in cellular activation.

Absence of terminal transferase may predict failure of remission induction in childhood ALL.

Two children with acute lymphoblastic leukemia (ALL), whose lymphoblasts lacked terminal deoxynucleotidyl transferase (TdT) by both enzyme and fluorescent antibody assay, responded poorly or not at all to vincristine and prednisone. Both patients had high presenting white counts and mixed L1-L2 morphology. Lymphoblasts from one patient, an adolescent boy with a mediastinal mass, possessed surface membrane receptors for sheep red cells (E) and for complement (EAC) and had elevated adenosine deaminase activity (ADA). Lymphoblasts from a 2.5-yr-old boy without a mediastinal mass did not form E or EAC rosettes and did not express the la-like antigen or carry surface immunoglobulin. The poor response to therapy and absence of TdT were associated with a lymphoblast phenotype suggestive of a highly differentiated T-cell-derived line in one instance and an undifferentiated cell in the other instance. It is postulated that absence of TdT may predict poor therapeutic efficacy of vincristine and prednisone in acute lymphoblastic leukemia in childhood. The absence of TdT may correlate with other developmental characteristics of lymphoblasts, such as altered function or low numbers of glucocorticoid receptors or resistance to lysis by steroid drugs. Determination of many parameters of lymphoblast phenotype at diagnosis to characterize the nature of the malignant cells more precisely may ultimately enhance our understanding of, and improve therapy for, the group of leukemic children who fail to respond to standard regimens.

Immunologic studies of cartilage-hair hypoplasia in the Amish.

The immunologic status of 18 Old Order Amish persons with cartilage-hair hypoplasia and 9 unaffected sibs was studied. Although none of the subjects had a history suggestive of persistent immune dysfunction, the subjects with cartilage-hair hypoplasia had significantly lower lymphocyte mitogenic and allogeneic cell stimulation responses when compared to unaffected sibs and unrelated control subjects. The abnormalities of cellular immune function found in the 18 affected subjects were similar to those reported in Finnish subjects with cartilage-hair hypoplasia.

An unusual form of measles meningoencephalitis. A report of two cases.

Two patients are reported with a chronic progressive illness characterized by dementia, ataxia and spasticity. There were no myoclonic jerks and both had normal electroencephalograms (EEG). Pathological findings in three brain biopsies were those of viral meningoencephalitis with perivenous demyelination. Serological data in both patients indicated the presence of measles virus infection. Intracytoplasmic structures resembling measles virus nucleocapsids were found in the brain biopsy of one patient. Immunofluorescent staining showed antibody in the temporal lobe biopsy of both patients. It is suggested that these patients are examples of a chronic form of measles meningoencephalitis hitherto undescribed.

The detection of human B lymphocytes by both light and electron microscopy utilizing colloidal gold labeled anti-immunoglobulin.

Colloidal gold was used to label rabbit anti-goat IgG for the indirect detection of goat anti-human IgD and IgM. Utilizing this technique, surface immunoglobulin was visualized on human blood B lymphocytes at both the light and electron microscopic levels of resolution. By this technique, percentages of lymphocytes were found to closely correlate with those of the standard immunofluorescent procedure. The ease of the procedure along with the clarity of the marker in both light and electron microscopy suggest wide applicability in clinical and research studies of lymphocyte membrane markers.

Passive transfer of delayed hypersensitivity to Blastomyces dermatitidis between mice.

This study further characterized the delayed hypersensitivity state induced in animals by Blastomyces dermatitidis exposure. Passive transfer of delayed hypersensitivity by transfer of cells and inhibition of migration of peritoneal exudate cells were studied, using sensitized mice of two inbred strains. Donor mice were subcutaneously inoculated with viable B. dermatitidis yeast cells. After 15 days, spleen cells or serum from these animals were injected intravenously into normal recipients of the same strain. After 24 h these mice were footpad tested with killed B. dermatitidis yeast cell antigen. Mice receiving spleen cells from sensitized animals had a significant increase in footpad thickness 24 to 48 h after testing. Those receiving only serum remained negative. Migration of peritoneal exudate cells from blastomyces-sensitive donor mice was inhibited by presence of blastomycin but not by mycobacterial antigen. Neither blastomyces-sensitive nor control animals reacted to footpad or migration inhibition testing with mycobacterial antigen.