Alenka Gagro

Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry

Acute RSV infection in infancy may produce some asthma‐like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type‐2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV‐infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL‐4 and interferon‐gamma (IFN‐γ) were measured by flow cytometry. The cells from RSV‐infected infants produced more IL‐4 and less IFN‐γ than those from healthy controls. IL‐4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL‐4 production was greatest in infants with mild infections, whereas IFN‐γ production increased with disease severity. Our conclusions are that RSV infection is associated with IL‐4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine‐producing cells.

Increased Toll-like receptor 4 expression in infants with respiratory syncytial virus bronchiolitis

The fusion protein of the respiratory syncytial virus (RSV) binds to the pattern recognition receptors, TLR4 and CD14, and initiates innate immunity response to the virus. The aim of the study was to investigate the expression of TLR4 on peripheral blood lymphocytes and monocytes in peripheral blood of infants in both acute and convalescent phase of RSV bronchiolitis (n = 26). In addition, TNF‐α expression in lipopolysaccharide‐stimulated monocytes was also assessed. The results showed TLR4 to be expressed predominantly by monocytes in both sick infants and controls. During the acute phase of infection monocytes up‐regulated TLR4 in eight infants, which returned to the levels recorded in controls 4–6 weeks from infection. There was no difference in the percentage of TNF‐α secreting monocytes. Of the clinical parameters tested, minimal oxygen saturation was found to correlate negatively with this expression in the group of infants with increased TLR4. Additional studies are under way to correlate this finding with the outcome of the immune response to RSV.

Effect of steroids on the frequency of regulatory T cells and expression of FOXP3 in a patient with systemic lupus erythematosus: a two-year follow-up.

Regulatory T cells (Tregs) maintain peripheral tolerance by restricting the activity of nearby immunocytes. The loss of tolerance can induce development of autoimmune diseases, such as systemic lupus erythematosus (SLE). It has previously been shown that SLE patients have lower levels of Tregs than controls. Herein we show that glucocorticoid therapy in a newly diagnosed SLE patient was accompanied with the increase in the percentage of CD4+CD25+Foxp3 T cells to the higher levels than in healthy controls. No effect of the therapy was observed on the percentage of CD4+CD25+ T cells. The observed difference in correlation of the CD4+CD25+ and CD4+CD25+Foxp3+ T cells levels with disease onset in a patient receiving steroids emphasized the importance of critical evaluation of Treg phenotype and their role in the control of SLE.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS)

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS‐positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double‐ and triple‐colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA‐DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, α2‐globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.

Expression of Lymphocytes FcεRII/CD23 in Allergic Children Undergoing Hyposensitization

Owing to the proposed role of Fc epsilon RII/CD23 in allergic diseases, we analyzed the expression of this receptor on peripheral blood lymphocytes (pan-B, pan-T and CD4+ or CD8+ T cells) and its autoproteolytic product sCD23 in serum. This was done in 10 asthmatic children allergic to Dermatophagoides pteronyssinus (Dpt) before and 6 weeks after hyposensitization. FACS analysis of double, direct immunofluorescence staining of the whole blood revealed an elevated percentage of Fc epsilon RII/CD23+ lymphocytes in allergic children (10.29 +/- 5.0), a significantly higher percentage than in nonallergic children (5.7 +/- 2.4, p < 0.05). The majority of Fc epsilon RII/CD23+ were on B cells. A significant positive correlation between the percentages of CD23+ lymphocytes and serum IgE levels was found (Spearman rank = 0.63, p < 0.05). The percentage of CD20+CD23+ lymphocytes significantly decreased after 6 weeks of hyposensitization (6.2 +/- 3.6, p < 0.05), while the percentage of CD20+ lymphocytes remained unchanged. Similarly, hyposensitization was followed by a reduction of total serum IgE levels, but Dpt-specific IgG4 and IgE remained unchanged.

Double Trouble: Hemorrhagic Fever with Renal Syndrome and Leptospirosis

The clinical picture of hemorrhagic fever with renal syndrome can closely mimic that of unicteric leptospirosis and vice versa. This is the first description of dual infection with Dobrava virus and leptospira and alteration of immune parameters in a Croatian soldier.

Type I cytokine profiles of human naïve and memory B lymphocytes: a potential for memory cells to impact polarization

B cells bifurcating along ‘type 1’ or ‘type 2’ pathways under the influence of polarizing cytokines can, in turn, influence the direction of an immune response. Here, we compare the capacity of human B cells residing within naïve and memory compartments to participate in type 1 polarizing responses. B‐cell receptor (BCR) engagement provided the main signal for interleukin (IL)‐12Rβ1 expression in the two subsets: this was potentiated by CD154 together with interferon‐γ (IFN‐γ) but inhibited by IL‐12. IL‐12Rβ2 could be induced on a minority of B cells by the same signals, and also by IFN‐γ alone. WSX‐1, a receptor for IL‐27, was expressed in both subsets with no evidence for its regulation by the signals studied. While neither subset was capable of secreting much IL‐12 p70, memory B cells could produce a small amount of IL‐12 p40 on CD40 ligation. Memory B cells also, exclusively, expressed IL‐23 p19 mRNA on BCR triggering. Importantly, products of appropriately stimulated memory – but not naive – B cells were shown to promote the synthesis of IFN‐γ in uncommitted T‐helper cells. The data indicate an equal capacity for naïve and memory B cells to respond within a type 1 polarizing environment. Although poorly equipped for initiating type 1 responses, B cells – by virtue of the memory subset – reveal a capacity for their maintenance and amplification following T‐dependent signalling.

Naive and memory B cells respond differentially to T-dependent signaling but display an equal potential for differentiation toward the centroblast-restricted CD77/globotriaosylceramide phenotype.

Resting (CD38low) tonsillar B cells differentiate to express the centroblast‐restricted CD77/globotriaosylceramide antigen on high‐level engagement of CD154. As the CD38low population comprises both naive and memory subsets, we wished to compare the propensity of each to develop this germinal center phenotype; particularly as the capacity of memory B cells to re‐enter afollicular reaction remains unclear. Resting B lymphocytes were therefore separated into CD27–IgA–IgG– and IgD– fractions to generate subsets enriched for naive and memory cells, respectively. Following stimulation via BCR and/or CD40 – surrogate signals for B cells engaged in T‐dependent signaling – differences between the two subsets were seen in the kinetics and/or magnitude of responses such as entry into DNA synthesis, induction of the costimulatory molecules CD80 and CD86; up‐regulation of CD23, and changes in BCL‐6 mRNA expression. Nevertheless, naive and memory cells revealed a nigh identical capacity for acquiring CD77: both appeared equally sensitive in this regard, with high‐level CD40 engagement via cell‐bound CD154 being required for both subsets to achieve the hallmark centroblast phenotype. These findings suggest that, provided with the opportunity to encounter cell membrane CD154 in abundance, both naive and memory B cells display the potential to be diverted towards a germinal center pathway of differentiation.

Effect of house dust mite immunotherapy on transforming growth factor β1-producing T cells in asthmatic children

BACKGROUND Recent evidence suggests that regulatory T cells (Treg cells) and immunosuppressive cytokines, such as transforming growth factor BETA1 (TGF-BETA1) and interleukin 10 (IL-10), may have a role in clinically effective allergen specific immunotherapy (SIT). OBJECTIVE To evaluate the effect of SIT on the induction of Treg cells in house dust mite-allergic children and on the expression of specific Treg cell markers (cytotoxic T-lymphocyte-associated protein 4 [CTLA-4], IL-10, and TGF-BETA1). METHODS In this uncontrolled open-label study, the percentage of peripheral blood CD4+ Treg cells (CD69 CD45RO+CTLA-4+ and CD3+CD4+CD25+FOXP3+) and the expression of molecules associated with their functions (CTLA-4, TGF-BETA1, and IL-10) were analyzed using flow cytometry in 16 children allergic to house dust mites before and at 3 and 12 months of subcutaneous SIT. Clinical variables, such as symptom score, medication requirements, forced expiratory volume in 1 second, peak expiratory flow rate, and serum IgE levels, were also determined. Ten healthy children were included as controls. RESULTS All the clinical variables improved during immunotherapy. The percentage of CD4+CD25+CD69-CD45RO+ Treg cells remained unchanged. The percentage of CTLA-4+ -expressing Treg cells transiently increased after 3 months of immunotherapy, whereas the percentage of FOXP3+ Treg cells did not change after 1 year of immunotherapy. Levels of IL-10+ cells transiently decreased after 3 months of immunotherapy. Four children who required inhaled fluticasone propionate administration for significant symptom worsening had no statistically significant increase in TGF-BETA1-secreting T cells at 12 months of SIT, in contrast to 12 children without inhaled corticosteroid treatment. CONCLUSIONS The increase in TGF-BETA1-positive T cells only in children without significant symptom worsening requiring inhaled corticosteroid treatment limits the usefulness of TGF-BETA1 in monitoring response to allergen immunotherapy.

Expression of CD23 antigen and its ligands in children with intrinsic and extrinsic asthma

The role of the low‐affinity IgE receptor CD23 in immune reactions has been further emphasized by recent discoveries of novel surface ligands for CD23: CD21, CD11b, and CD11c. We previously observed the difference between the expression of CD23 and CD21 antigens in children suffering from extrinsic asthma when compared to healthy controls. In the present study, we investigated the expression of CD23 and its ligand CD21 on CD20+B cells in 44 asthmatic children (23 allergic and 21 nonallergic) using three‐color immunofluorescence analysis. In addition, the expression of two other ligands for CD23, CD11b, and CD11c, on T cells (CD3+), a subpopulation of T cells (CD4+ and CD8+), natural killer cells (CD56+), and monocytes (CD14+) was tested by two‐color immunofluorescence analysis in 12 allergic and 14 nonallergic children. We found that children with extrinsic asthma had higher levels of CD23+ B cells than those with intrinsic asthma. No difference was observed in the percentage of either CD23+CD21+ or CD23‐ CD21+ B cells. The CD11b antigen was expressed on each tested population, but only on CD4+ T cells was CD11b significantly increased in children with extrinsic asthma. CD11c was expressed mainly on monocytes, and no difference was observed between tested groups. The increased percentage of CD11b antigen on CD4+ T cells and the increased percentage of CD23 antigen on B cells in children with extrinsic asthma provide further evidence of the immunologic differences between intrinsic and extrinsic asthma.