Evidence that the c, D and E epitopes of the human Rh blood group system are on separate polypeptide molecules.

The distribution of the epitopes, c, D, E and G of the human Rh system on red cells has been investigated using both 125I-labelled and fluorescently-labelled MAbs. There is a very wide range in the density of each epitope on individual red cells and the numbers of c, D and E epitopes on each cell are independent of each other. These observations taken together with the finding that there is no steric hindrance to binding between the antibodies, anti-c, anti-D and anti-E indicate that these epitopes are on separate Rh polypeptide molecules. The observations are taken to indicate that the total amount of Rh polypeptide on a single red cell is constant but that there is considerable heterogeneity between cells in the amount of each separate polypeptide carrying the different epitopes. In contrast, there was a mutual inhibition of binding of anti-G and anti-D monoclonals and direct positive correlation between the number of G and D sites on individual cells, indicating that the G and D epitopes are probably on the same Rh polypeptide.

Progression to AIDS in the majority of asymptomatic HIV-infected people.

Sixty-eight asymptomatic HIV-seropositive people with a CD4 lymphocyte count above 400/mm3 at the first examination were followed up every year over a 3-year period, by monitoring the biological markers of AIDS (CD4 lymphocyte decrease, loss of anti-p24 or anti-p17 antibodies, positive p24 antigenemia, increase of erythrocyte sedimentation rate, and of serum levels of immunoglobulin G. immunoglobulin A, neopterin and beta 2-microglobulin). The percentages of subjects positive for at least one marker at the first, second, third and fourth examinations were 66, 88, 94 and 97%, respectively. The increase in the number of markers with time was significant (chi-square test; P less than 0.001). This increase suggests a progression to AIDS in the majority of asymptomatic seropositive subjects, even those without a decreased CD4 lymphocyte count.

Disappearance of CD4-lymphocyte circadian cycles in HIV-infected patients: early event during asymptomatic infection.

Circadian variations have been observed in peripheral blood lymphocyte counts in normal subjects; they usually reflect variations in absolute CD4-cell count. For this population, nadir occurs around 0800 h (basal value) and the peak value occurs at midnight (1.6 times the 0800-h value). This cycle is thought to be of major importance in the efficacy of the immune response because it expresses the migration of lymphocytes into lymphoid organs.

Frequency and prognostic importance of thrombocytopenia in symptom-free HIV-infected individuals: a 5-year prospective study.

The exact frequency of HIV-associated thrombocytopenia (TCP), defined as platelet count less than 150 x 10(9)/1, was studied in 435 symptom-free HIV-seropositive individuals. At the baseline control, 23 (5.5%) had TCP. TCP individuals had a significantly lower mean CD4 lymphocyte count than the non-TCP individuals. During a mean follow-up of 30 months, 79 out of the 435 individuals (18%) had TCP at least once. During the study period, only 1% of our patients had a platelet count less than 50 x 10(9)/l. TCP was more frequent in intravenous drug users than in other risk groups. A spontaneous normalization of platelet count was observed in more than 50% of TCP individuals.

Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV.

Systematic screening of blood donations by enzyme-linked immunosorbent assay (ELISA) for HIV antibodies carries a false-positive rate: the sera involved react in Western blot to core antigens (p24 or p17) but reactivity to envelope is absent. We studied 22 subjects with persistent and isolated anti-core reactivities; 75 HIV seropositive patients were controls. The epidemiological data and the follow-up and biological tests performed in these two populations argue that donors with persistent and isolated anti-core antibodies are not seroconverting for HIV. We conclude: (1) that verification of all anti-HIV ELISA-positive sera by Western blot is essential and that the presence of at least once anti-envelope (gp120 or gp41) antibody is indispensable for the diagnosis of HIV infection; (2) that the solitary anti-p24 or anti-p17 bands observed on Western blot are false-positive. There is no evidence that donors with such reactivities are HIV-infected.

Circadian variations in plasma levels of hypophyseal, adrenocortical and testicular hormones in men infected with human immunodeficiency virus.

Alterations in the circadian time structure of the secretion of several hormones were investigated in 13 male patients infected with human immunodeficiency virus (HIV). Seven were asymptomatic (classified CDC II, according to the criteria of the Atlanta Centers for Disease Control), and 6 had acquired immunodeficiency syndrome (CDC IV). Ten healthy males volunteered as controls. Plasma levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), cortisol, testosterone, ACTH, and beta-endorphin were determined by RIA in blood samples obtained every 4 h from 0830-0830 h the next morning. Data were analyzed both by two-way analysis of variance and the cosinor method. Circadian rhythms were statistically validated for each of the six hormones in each of the three groups of subjects. Compared with the control subjects, mesors (24-h adjusted means) were significantly higher for cortisol and lower for DHEA, DHEA-S, and ACTH (P less than 0.001 for all four hormones) in all HIV-infected patients. Plasma testosterone mesors were similar in controls and CDC II patients, but decreased significantly in the CDC IV patient group (P less than 0.05). Analysis of the circadian rhythms of plasma hormone levels clearly indicated an altered adrenal hormonal state in HIV-infected male patients, even during the asymptomatic period of the infection. For instance, plasma cortisol at 0430 h was more than twice as high in HIV-infected patients as it was in time-qualified controls. Although patients already had elevated plasma cortisol and lowered adrenal androgen levels at this stage, hypogonadism was not observed, as gauged by plasma testosterone concentrations. We speculate that the primary hormonal defect in HIV-infected patients is increased cortisol secretion resulting from circadian-varying stimulation of the adrenal cortex by a factor other than pituitary ACTH. This factor might be a stimulating substance secreted primarily by infected immune cells. Excess cortisol would lower adrenal androgen secretion by shifting adrenal steroid biosynthesis toward glucocorticoids and decreasing pituitary ACTH secretion via a negative feedback mechanism.

Clinical and biological features in the 12 months preceding onset of AIDS in HIV-infected subjects.

Biological markers of HIV infection were studied in 17 asymptomatic HIV seropositive subjects in the 12 months preceding the onset of the disease. No single marker of HIV infection preceded the development of AIDS. Therefore, the clinical care of asymptomatic seropositive subjects should include a number of tests to evaluate HIV activity and immune suppression.

Radioactive labeling techniques for the identification of G antigen in the human Rh blood group system.

The G antigen is one of the erythrocyte membrane Rh antigens. The amount of Rh antigen present on the red blood cell is about 10(-15) g and radioactive labeling of membrane proteins is a useful method for its identification and characterization. In this paper, we compare 4 labeling techniques. Using a human monoclonal anti-Rh(G) antibody and an immunofixation technique, we located the G antigen on a polypeptide of an average molecular weight of 28,000 Da.

A rapid technique for lymphocyte preparation prior to two-color immunofluorescence analysis of lymphocyte subsets using flow cytometry. Comparison with density gradient separation.

A technique is described for lymphocyte preparation which permits analyses by two-color immunofluorescence and flow cytometry. It consists, briefly, of the lysis of red blood cells and washing of white blood cells prior to labeling. We tested this technique with a large panel of monoclonal antibodies in mono- and dual immunofluorescence. By comparing these results to those obtained after density gradient separation, we found the following statistically significant differences: the count of the phenotype B1+ was higher after whole blood lysis preparation than after density gradient separation; whereas, the corresponding counts of OKT4+ and Leu-4-Leu-7+ phenotypes were lower. No difference was detected with OKT8+, Leu-4+, OKT8+Leu-4+, OKT8+Leu-4-, OKT8-Leu-4+, OKT8+Leu-7+, Leu-4+Leu-7+, Leu-4-Leu-11c+, OKT8+Leu-11c+ and OKT8+Leu-15+ phenotypes. We have studied the reproducibility of both methods and the correlation between them. The disparity of the lymphocyte subset count between these two methods, though statistically significant, was relatively weak and seems to be due to the density gradient separation. Since the preparation of lymphocytes using the density gradient method is time consuming, we propose whole blood lysis as an alternative lymphocyte separation method when assessing immune status in human disease by flow cytometry. It offers the following advantages: (i) it does not require additional steps, (ii) it permits two-color immunofluorescence through the labeling of white blood cells after washing, (iii) it is reliable, (iv) it is reproducible, and (v) it is helpful in studies of lymphopenia since it offers the possibility of lymphocyte enrichment.

CD16+ NK cells decrease in all stages of HIV infection through a selective depletion of the CD16+CD8+CD3- subset.

Natural killer (NK) cell-related phenotypes were analyzed in human immunodeficiency virus (HIV) infection. Our study involved 168 HIV-infected patients (72 CDC Stage II, 48 Stage III, and 46 Stage IV) and 60 healthy individuals. Analyses were conducted using flow cytometry and monoclonal antibodies. In comparison to the control group, all patient groups showed a significant decrease (p less than 0.001) of the CD16+ and CD16+CD3- phenotypes; furthermore, the comparison among patient groups showed no significant difference. It seems, therefore, that the decrease begins in the asymptomatic stage (CDC II) and remains constant during the infection. CD16+ NK cells were further divided into two subsets: CD16+CD8+ and CD16+CD8-. This subdivision shows a severe selective depletion of the CD16+CD8+ subset, but not in the CD16+CD8- subset. The depletion of the CD16+CD8+ subset also appears in the CDC asymptomatic stage and remains constant in CDC stages III and IV. Elsewhere, we observed that CD16+CD8+ lymphocytes are CD3-; complementary analysis of CD3-CD8+ cells showed a depletion comparable to that of the CD16+CD8+ phenotype. Depletion of the CD3-CD8+ subset, which belongs to the NK cell compartment, was observed although the total CD8 population showed a statistically significant increase. We conclude that, in HIV infection, there is a quantitative decrease of the NK CD16+ cell population, which appears to be due to a selective depletion of the CD16+CD8+CD3- compartment. This severe depletion appears to begin early in the infection.

Human brain lectin immunoreactive material in cerebrospinal fluids determined by enzyme immunoassay (EIA).

A sensitive enzyme immunoassay (EIA) micromethod is described which can measure levels of a 14 kDa human brain lectin (HBL) in the cerebrospinal fluid (CSF) of patients submitted to CSF examination. The assay is based on the use of a polyclonal antibody to HBL and the simultaneous application of biotinylated and unlabeled HBL. Biotin was then reacted with a streptavidin-peroxidase (Strep-HRP) conjugate and the bound enzyme quantified with the substrate orthophenylenediamine (OPD). The assay requires only 50 microliters of CSF and is very sensitive: as little as 6 ng/ml of HBL 14 can be detected. In a blind-test screening, the mean (+/- SEM) concentration of the HBL immunoreactive material (HIM) in CSF was determined to be 72.4 +/- 6.6 ng/ml. Our results indicate that EIA measurement of HIM levels in the CSF may find useful applications in elucidating the involvement of HBL in the physiopathology of human nervous system (NS).

Disappearance of CD4 lymphocyte circadian cycles after autologous bone marrow transplantation.

Circadian variations are observed in CD4 lymphocyte count in healthy people. Samples taken of the basal value occurring around 08.00 hr and peak value at midnight made it possible to define the range of cyclic variations. Movement of lymphocytes seems important in accounting for the observed circadian variations. CD4 circadian cycle amplitudes were investigated in 12 patients with autologous bone marrow transplantation. Cycle abnormalities were observed in 7 patients. Two of them had a normal CD4 lymphocyte count. It was therefore possible for functional abnormalities in CD4 lymphocytes to be detected in patients with a normal CD4 count. Because of these results, we are of the opinion that the evaluation of CD4 lymphocyte cycle might be a more sensitive test than the absolute CD4 count itself.

Circadian rhythms of circulating NK cells in healthy and human immunodeficiency virus-infected men.

Antiviral immunity involves NK cells, which circulate rhythmically every 24 hours. We have investigated circadian and 12-hour rhythms in the peripheral count of circulating NK cells in 15 men infected with human immunodeficiency virus (HIV) and 13 healthy controls. We analyzed three phenotypes using double-labeling with monoclonal antibodies and flow cytometry assessment: CD3- CD16+, CD3-CD57+, and CD2+CD3-. A statistical validation of time-dependent differences was achieved if significance (p < 0.05) was validated both with analysis of variance and cosinor. The circadian rhythm had a similar asymmetric waveform for the three phenotypes and is homogeneous on an individual basis. The circulating NK cell count peaked in the early morning and was low at night. A circadian rhythm and a circahemidian harmonic characterized all phenotypes in healthy subjects. We considered two groups of HIV-infected men: those who were asymptomatic (eight) and those with acquired immune deficiency syndrome (AIDS) (seven). Circadian changes in NK cell count were similar in both subgroups and in healthy controls. The circadian pattern was also consistent among individual patients. Asymptomatic HIV-infected men (early-stage disease) exhibited more pronounced 12-hour rhythmicity than did patients with AIDS or controls. The circulation of NK cells does not appear to share the same synchronizer(s) as other circulating T- or B-lymphocyte subsets. Thus, HIV infection gradually abolished circadian rhythmicity in circulating T and B cells, whereas it did not disturb that in NK cells.

[Early anomalies of CD4 and CD20 lymphocyte cycles in human immunodeficiency virus]. / Anomalies précoces des cycles lymphocytaires CD4 et CD20 au cours de l'infection par le virus de l'immunodéficience humaine.

Circadian variations in the number of circulating lymphocytes and their subpopulations have been observed in healthy subjects. These cyclic changes are characterized by a trough at 8:00 a.m. and a peak at midnight. Using multiple peripheral blood samplings, we were able to confirm that this cycle applied to CD4 T-cells (helpers) and to B-cells (CD20). No cycle of CD8 lymphocytes was observed. In a second stage, for greater comfort of the patient the number of samplings was reduced to two: one at 8:00 a.m. (trough) and one at midnight (peak). This method enabled us to calculate the amplitude of lymphocytes cycles in 18 controls and 74 human immunodeficiency virus (HIV) seropositive patients. In asymptomatic HIV carriers the amplitude of CD4 cycles was normal in 6/26 cases and that of B-cell cycles in 2/17 cases. In the group of asymptomatic HIV carriers the mean amplitude of the cycles was much less reduced than in the other two groups. These results incite us to believe that the loss of the CD4 T-cell cycles is an early sign of HIV infection antedating the decrease observed in the number of these cells.

[I antigenicity of the Bombay red cells]. / Antigénicité I des hématies Bombay

Different homogeneous IgM cold agglutinins have been used to determine the antigen site density of Oh and OHm erythrocytes in comparison with O (H). The equilibrium constants and the delta Ho and delta S o have been measured. When H antigen is absent on the O red blood cells, there is no apparition of new I and i antigenic sites. Competition between gene products converting i into I and Hh genes for a common precursor substance appears unlikely. Nevertheless, the affinity of all antibodies tested increases with Oh and OHm cells and the thermodynamic parameters are accordingly modified. These results could be explained by: either a lesser steric hindrance in the absence of H antigen, the I determinants that are normally hidden by H antigen become more accessible, or by a modification of the molecular environment, as the interaction between fucosyl and N-acetyl residue of the penultimate sugar on the basic oligo-saccharidic chain is abolished. The structure of some I determinants may be modified, particularly of those which include the beta-D-Glu-NAc. Another explanation for these peculiar reactivities could be the substitution on the Bombay cells of this fucose by a neuraminyl residue. In the Sch. siblings, a heterozygous Hh subject was evidenced. His thermodynamic characteristics are nearing those of the hh propositus; the hypothesis of a competition between fucosyl and neuraminyltransferases for the same substrate is then proposed.

[Flow cytometry in immunology and hematology: some essential practical aspects]. / La cytométrie en flux en immunologie et en hématologie: quelques aspects pratiques essentiels.

Flow cytometry, supported by monoclonal antibodies, has widely contributed in the cellular identification, notably in the clinical and haematological fields. This technique has found several applications since the qualitative phenotyping and the quantitative analysis of the immune system's cell populations are helpful in the diagnosis and the therapy. Besides, these indications require an appropriate knowledge of several methodological aspects including factors related to the sample donor: age and sex; sampling: time and quantity; and the sample preparation conditions. References values, needed for the results interpretation, have a meaning only if they are defined within these validity limits. Previous trials have been done in order to define a biological value representative of the immunological status, such as the CD4/CD8 ratio. Unfortunately this ratio is not justified in the scope of new knowledge concerning the cellular interactions and the functional heterogeneity of cells involved in the immune system.

Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites. Quantitative and thermodynamic studies.

The effects of neuraminidase and proteolytic enzymes on I and i reactivities was studied with I and i adult red cells, using radioimmunological methods. An enhanced reactivity after enzyme treatment is not exclusively due to a membrane charge reduction. The increase in site numbers and association constants bring about the gain of the cold agglutinin fixation. The release of N-acetylneuraminic acid residues gradually increases the I antigen site density of I red cells and the i site density of i red cells. Similar behaviour was observed after proteolytic enzyme treatment with papain, bromelin or ficin. The proteolytic treatment of I erythrocyte reveals underlying i receptors on these cells. Following membrane glycoprotein chain removal, anti-i antibodies are specifically fixed on I erythrocytes. The accessibility to antibodies of the determinants responsible for I and i erythrocyte activities was influenced significantly by steric hindrance factors. While N-acetylneuraminic acid release increased antibody affinities for the antigenic receptor, the removal of glycopeptide chains greatly diminished steric hindrance and brought about higher affinity constants. After enzyme treatment, the antigenic structures become more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane.