Nora Galassi

Diclofenac induces basophil degranulation without increasing CD63 expression in sensitive patients

Diclofenac (Dc) induces an IgE‐independent basophil (Ba) degranulation in susceptible individuals. CD63 Ba expression is utilized as an in vitro test for diagnosis of drug hypersensitivity. We tested the ability of Dc to induce CD63 Ba expression by flow cytometry (BAT) and Ba degranulation using light microscopy (HBDT) in patients sensitive to Dc. We studied 14 patients with diclofenac hypersensitivity, also two patients sensitive to Dermatophagoides pteronyssinus (Dp), and 12 normal controls. HBDT was performed by mononuclear cells toluidine blue staining. BAT determined CD63 expression in antiCD63/anti‐IgE/anti‐CD45‐labelled whole blood. In each case, the percentage of activated Ba post‐stimulation with 1 and 10 µg/ml Dc was determined. Positive controls included N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) peptide‐induced activation. IgE‐mediated Ba activation was induced with a Dp allergenic extract. With Dc 1 µg/ml, mean HBDT in Dc‐susceptible individuals was 33·62 ± 18·35% and 8·49 ± 4·79% in controls (P = 0·0001). Mean BAT was 2·04 ± 1·68% and 1·93 ± 1·40% in controls (P = 0·8). Ba preincubation with Dc did not affect fMLP‐induced CD63 expression, neither in Dc‐sensitive individuals (P = 0·8) (n = 4) nor in subjects without Dc hypersensitivity (P = 0·25) (n = 4). Ba from the two patients sensitive both to Dc and Dp responded to Dp but not to Dc by BAT: Dc, 1·99 ± 0·78%; Dp: 60·87 ± 9·28%; but showed degranulation by HBDT: Dc, 30·53 ± 1·02%, Dp: 48·78 ± 22·17%. Dc induces Ba degranulation in sensitive patients in a way that does not induce CD63 expression and is different from IgE‐mediated and fMLP‐mediated degranulation. Our results suggest that CD63 expression is not a reliable diagnostic method for diclofenac allergy.

Anti-leukocyte antibodies as a consequence of HIV infection in HIV+ individuals

Antibodies (Ab) that induce antibody-dependent cell-mediated cytotoxicity (ADCC) against non-T lymphocytes, anti-HLA class II specific Ab and anti-PMN were tested in hemophilic (He) patients who were alloimmunized because they had received replacement treatment with blood derivates and become infected with HIV, as well as in those who remained seronegative. In addition, the serum reactivity of spouses of HIV+ individuals and their children was studied to determine the effect of HIV infection in the absence of concomitant alloimmunization. The results of this study indicate that ADCC Ab were already present in HIV- He, suggesting the influence of alloimmunization. Their titer increased after appearance of HIV disease. While low reactivity against class II antigens was observed in HIV- He, activity augmented sharply after HIV infection and increased further with disease progression. Anti-PMN reactivity followed a similar pattern. Anti-class II, ADCC Ab and anti-PMN were also detected in the asymptomatic HIV+ spouses of HIV+ patients in titers that were similar to those of asymptomatic HIV+ He. In children born to HIV+ mothers in whom HIV infection was confirmed, anti-class II, ADCC Ab and anti-PMN reactivity were also observed, and activity increased after the onset of disease. These results suggest that induction of anti-leukocyte Ab occurs in the absence of massive allostimulation after HIV infection. HIV infection may enhance preexisting class II and anti-leukocyte response in allostimulated individuals.

A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay

Summary.  In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme‐linked immunosorbent assay (ELISA) that detects both inhibitory (I‐Ab) and non‐inhibitory (NI‐Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I‐Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII‐m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control‐m). Captured anti‐FVIII antibodies were detected using anti‐human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I‐Ab, <0.5 BU mL−1); 13 PI (patients with I‐Ab, 1.1–8200 BU mL−1). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII‐m and control‐m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI‐Ab by FC, and two of them developed high levels of I‐Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.

Leukoagglutinins in patients with hemophilia

Anti-leukocyte antibodies may occur in hemophilic patients as a consequence of replacement therapy with blood derivates. In this report we describe the presence of leukoagglutinins (LA) in serum of HIV-infected hemophiliacs (HIV + He) and their absence in HIV-negative patients (HIV-He). LA activity was recovered in IgG fractions from HIV + He, in the polyethylene glycol (PEG) precipitates from these sera, and in some cases in the supernatant fractions of PEG precipitates. Although the amount of PEG precipitates corresponding to circulating immune complexes (CIC) was higher in HIV + He than in HIV-He and normals, there was no direct relationship between CIC levels and LA. LA reacted both with autologous and with allogeneic polymorphonuclear leukocytes (PMN). In contrast to PMN isolated from HIV-He, HIV + He PMN had membrane associated IgG. In HIV + He, LA activity was more frequently observed in patients with more advanced stages of HIV infection than in asymptomatic individuals. Our results suggest that LA activity could be one of the manifestations of autoreactivity associated with progressing HIV infection in patients with hemophilia.

Common Variable Immunodeficiency and Circulating TFH

CD4+ T follicular helper cells (TFH) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). TFH lymphocytes were characterized by expression of CXCR5 and PD-1. TFH were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and TFH reg were similar in both CVID groups and in N. TFH responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of TFH cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that TFH are functional in CVID and highlight the association of increased circulating TFH with AI and GD manifestations.

Inhibition of normal natural killer cytotoxicity by sera from hemophilic patients

In this study we searched for circulating antibodies or other serum factors that could account for the natural killer (NK) defect observed in hemophiliacs (He) infected with the human immunodeficiency virus (HIV). We analyzed the effect of negative or positive sera for HIV from He on normal NK activity. We showed that sera from He interfered with normal NK cytotoxicity. The inhibitory activity was higher in HIV+ sera and increased as the HIV disease progressed. HIV- sera also inhibited NK function, although to a lesser extent than HIV+, and it was probably due to isoimmunization through replacement treatment with plasma-derived concentrates. For each individual, no direct correlation was found between NK inhibition (NK-INH) of sera and the NK activity of He peripheral blood mononuclear cells (PBMC). Furthermore, He serum was poorly inhibitory on autologous PBMC. Preincubation of allogenic effector or target cells with He sera revealed that the inhibitory effect was the result of the reaction with these cells. A positive correlation was found by comparing NK-INH of whole He sera with the serum levels of circulating immune complexes. When the NK-INH assay was performed using the same concentration of DEAE-purified IgG from N, HIV- or HIV+, we found that HIV+ AIDS IgG was more inhibitory than the others.

Immune response to Streptococcus pneumoniae in asthma patients: comparison between stable situation and exacerbation.

In Argentina, more than 3 million people suffer from asthma, with numbers rising. When asthma patients acquire viral infections which, in turn, trigger the asthmatic response, they may develop subsequent bacterial infections, mainly by Streptococcus (S.) pneumoniae. This encapsulated Gram+ bacterium has been considered historically a T cell‐independent antigen. Nevertheless, several papers describe the role of T cells in the immune response to S. pneumoniae. We evaluated the response to S. pneumoniae and compared it to the response to Mycobacterium (M.) tuberculosis, a different type of bacterium that requires a T helper type 1 (Th1) response, in cells from atopic asthmatic children, to compare parameters for the same individual under exacerbation and in a stable situation whenever possible. We studied asthma patients and a control group of age‐matched children, evaluating cell populations, activation markers and cytokine production by flow cytometry, and cytokine concentration in serum and cell culture supernatants by enzyme‐linked immunosorbent assay (ELISA). No differences were observed in γδ T cells for the same patient in either situation, and a tendency to lower percentages of CD4+CD25hi T cells was observed under stability.

Severe haemophilia A patients have reduced numbers of peripheral memory B cells.

Summary.  The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII‐specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti‐FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti‐FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non‐switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti‐FVIII response and to monitor the success of ITI.

Diethyltoluamide (DEET) increases CD63 expression in a contact urticaria patients basophils.

Fil: Galassi, Nora Virginia. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Increased CD4-positive monocytes in HIV-infected haemophilic patients

The monocyte–macrophage system is known to play a central role in HIV infection, and expression of CD4 on the surface of monocytes/macrophages is important, since this molecule is a key factor for the entrance of HIV into susceptible cells. In this paper we evaluated the expression of CD4 in monocytes of haemophiliac patients (He) who had been infected with HIV (HIV+ He) through transfusion of contaminated plasma concentrates. Thirty seropositive patients (HIV+ He), 10 seronegative He patients (HIV− He) and 20 voluntary normal blood donors were studied. Phenotypic evaluation of monocytes was performed by flow cytometry of peripheral blood stained with anti‐CD45, ‐CD3, ‐CD4 and ‐CD14 monoclonal antibodies. The percentage of CD4 monocytes was increased in all HIV+ patient groups, but it was highest in those belonging to Groups III and IV A of the CDC classification. Furthermore, the median of fluorescence intensity of CD4+ monocytes from individual patients was shifted to the right, indicating expression of increased numbers of CD4 molecules on the cell membrane of monocytes. This could in turn favour HIV infection and viral persistence, facilitating in vivo dissemination of the virus.