Clinical and Experimental Immunology

We have previously reported the presence of marked immune dysregulation with a dominant Th2 profile, in a population of Ethiopian immigrants (ETH) in Israel heavily infected with helminths. In order to characterize better this immune dysregulation we studied by flow cytometry the expression of several activation markers on peripheral T cell populations, and lymphocyte apoptosis, in blood samples obtained from 63 ‘new’ ETH (recently arrived), 18 ‘old’ ETH (> 5 years since immigration) and 34 non-Ethiopian Israelis. The main findings in the ‘new’ ETH group in comparison with the non-Ethiopian controls were: (i) decreased CD4 and increased CD8 lymphocyte counts; (ii) elevated levels of activated T cells (CD3, CD4 and CD8) expressing HLA-DR; (iii) decreased levels of ‘naive’ CD4+ cells (CD45RA+), with increased levels of ‘memory’ CD4+ cells (CD45RO+); (iv) decreased numbers of CD28+ CD8+ lymphocytes; (v) marked increase in lymphocyte apoptosis. These T cell alterations and activation profile remained unchanged in 10 ‘new’ ETH in whom the helminth infections persisted for 6–11 months. In contrast, in 18 ‘old’ ETH, without helminth infections, the T cell activation profile was within the normal range. These findings suggest that chronic helminth infections may have a profound effect on the immune system of the host that disappears after eradication of these infections and adjustment to the new environment. It should therefore be taken into consideration for every immunomodulation therapy and especially in vaccine design and trials, in regions endemic for helminth infections.

Các người nhận ghép tạng bị nhiễm virus cytomegalovirus (CMV) có tỷ lệ tế bào lympho CD8+ tuần hoàn tăng lên. Một nghiên cứu theo chiều dọc trên 11 người nhận ghép thận và 5 người nhận ghép gan có nhiễm CMV nguyên phát nhưng không có yếu tố nguyên nhân nào khác gây ra rối loạn chức năng ghép cho thấy sự mất cân bằng chọn lọc của các phân nhóm T tế bào CD8+ trong máu ngoại biên. Ban đầu, sự nhiễm CMV có viremia đi kèm với sự gia tăng số lượng T tế bào CD8+ sáng và hoạt hóa T tế bào. Các dấu hiệu hoạt hóa trở về mức bình thường khi nuôi cấy virus trở thành âm tính (trước khi kết thúc tháng đầu tiên). Trong khoảng thời gian từ tháng thứ hai đến tháng thứ sáu, đa số (12/16) bệnh nhân duy trì số lượng T tế bào CD8+ cao (1050-2900 tế bào CD8+/mm3), bao gồm một phân nhóm tế bào CD8+ không phổ biến, khi 45-73% lympho CD8+ sáng là CD3+ và TCRαβ+, nhưng không bị nhuộm bởi kháng thể anti-CD28, CDIIb, CD16, CD56 và CD57. Đáng ngạc nhiên là, tế bào T CD8+CD57+, một dấu hiệu đặc trưng của nhiễm CMV, không xuất hiện cho đến tháng thứ hai đến tháng thứ sáu của nhiễm CMV nguyên phát, và số lượng của chúng tăng dần sau đó. Chúng trở thành phân nhóm tế bào T CD8+ chủ yếu sau 6 tháng nhiễm bệnh và sự tồn tại của chúng trong vài (lên đến 4) năm có mối tương quan mạnh (r = 0-87) với sự mở rộng của các tế bào CD8+. Qua phân tích bằng MoAbs, không có ưu thế nào đối với việc sử dụng các gia đình gen TCR-Vβ cụ thể vào bất kỳ thời điểm nào trong nhiễm CMV nguyên phát. Do đó, sự tồn tại của tình trạng lymphocyt CD8 là mối liên hệ trực tiếp với tỷ lệ mở rộng của một phân nhóm CD8+ CD57- không phổ biến và sự thay thế tiến triển của nó bằng các tế bào T CD8+ CD57+ được kích thích mạn tính bởi CMV.

Previous studies using in vitro systems with various stimuli have shown that PBMC from patients with AD show increased levels of IL-4 but decreased levels of interferon-gamma (IFN-γ) compared with PBMC from normal controls. However, in vitro conditions do not always mimic the in vivo condition. We therefore believe that it is important to quantify the expression of these cytokines in freshly isolated PBMC. This study examines the expression of IFN-γ, IL-4 and IL-13 mRNA in freshly isolated PBMC from adult patients with AD, from patients with psoriasis vulgaris and from healthy adults, using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Levels of IFN-γ mRNA were significantly lower in PBMC of patients with AD than in controls. IL-4 mRNA levels did not differ significantly between groups. Conversely, levels of mRNA for IL-13 were significantly greater in PBMC of patients with AD than in controls. An increase in IL-13 expression may regulate the in vivo synthesis of IgE in patients with AD.

T helper type 9 (Th9) cells are a novel identified subset of CD4+ T helper cells, which could partly contribute to allergic inflammation, while the precise contribution of Th9 cells in atopic dermatitis (AD) remains unknown. We aimed to explore the possible role of Th9 cells in AD pathogenesis. The Th9 cell percentage, transcription factor PU.1 and cytokine interleukin (IL)-9 mRNA levels, as well as IL-9 serum concentration in peripheral circulation, were measured in AD patients, psoriasis patients and healthy controls. The Th9 cell percentage, PU.1 and IL-9 expression levels of AD patients were all increased significantly compared with the other two control groups (P < 0·01), and correlated positively with SCORing Atopic Dermatitis index, serum immunoglobulin (Ig)E and thymus- and activation-regulated chemokine (TARC) levels (P < 0·05). In simple AD patients and AD patients complicated by allergic rhinitis or asthma, there were no significant differences in the Th9 cell percentage, PU.1 and IL-9 expression levels between them. At the same time, IL-9 and vascular endothelial growth factor (VEGF) mRNA levels were detected in AD lesions and normal skin samples, which were both distinctly elevated in AD lesions, and there was a positive association between them (P < 0·01). Keratinocytes were cultured with IL-9 stimulation and the secretion of VEGF was detected. IL-9 can promote the secretion of VEGF by keratinocytes in a time- and dose-dependent manner. In conclusion, the expansion of the Th9 cell subset, up-regulation of the PU.1 transcription factor and increased secretion of the IL-9 cytokine may contribute to the pathogenesis of AD, which may be supported by the increased release of VEGF by keratinocyes after IL-9 stimulation.

An imbalance of interferon-gamma (IFN-γ)-bearing CD4+ T (Th1) cells in the pathogenesis of AD is well recognized; however, a possible role in AD for CD8+ T cells secreting Th1-like cytokines (Tc1) has not been properly addressed. In this study, two- and three-colour FACS analysis allowed us to discriminate the Th1 from the Tc1 subset. AD patients had half the number of IFN-γ-producing circulating T cells (P < 0.005; 13.6 ± 1.9% (mean ± s.d.)) compared with normal donors (25.0 ± 2.4%). Specifically, both Th1 (4.8 ± 0.7%) and Tc1 (8.1 ± 1.1%) cells in AD were decreased compared with Th1 (8.8 ± 0.8%) and Tc1 (15.0 ± 1.5%) cells in controls. Moreover, at the mRNA level, the ratios of IFN-γ/IL-4 and IFN-γ/IL-10 were lower in cells from AD patients compared with controls. In conclusion, the decrease of IFN-γ-producing T lymphocytes in AD is due to a reduction in both Th1 and Tc1 IFN-γ-secreting cells; this may not only contribute to the over-production of IgE, but also explain the high incidence of cutaneous infections observed in AD patients.

Ankylosing spondylitis (AS) is a complex genetic disease in which both MHC and non-MHC genes determine disease susceptibility. To determine whether the T cell repertoires of individuals with AS show signs of increased stimulation by exogenous antigens, CD4+ and CD8+ T cell subsets of five monozygotic HLA-B27+ twins (two concordant and three discordant for AS) and CD8+ T cell repertoires of three healthy HLA-B27+ individuals were characterized by TCR β-chain (TCRB) CDR3 size spectratyping. Selected TCRB-CDR3 spectra were further analysed by BJ-segment analysis and TCRB-CDR3 from expanded T cell clones were sequenced. In an analysis of all data (519/598 possible TCRB-CDR3 spectra), AS was associated with increased T cell oligoclonality in both CD8+ (P = 0·0001) and CD4+ (P = 0·033) T cell subsets. This was also evident when data were compared between individual twins. Nucleotide sequence analysis of expanded CD8+ or CD4+ T cell clones did not show selection for particular TCRB-CDR3 amino acid sequence motifs but displayed sequence homologies with published sequences from intra-epithelial lymphocytes or synovial T cells from rheumatoid arthritis patients. Together, these results provide support for the hypothesis that responses to T cell-stimulating exogenous or endogenous antigens are involved in the induction and/or maintenance of AS.

The pattern-recognition molecules mannan-binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL-associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an important component of the innate immune system. Three proteins are produced from the MASP1 gene: MASP-1 and MASP-3 and MAp44. We present an assay specific for MASP-1, which is based on inhibition of the binding of anti-MASP-1-specific antibody to MASP-1 domains coated onto microtitre wells. MASP-1 was found in serum in large complexes eluting in a position corresponding to ∼600 kDa after gel permeation chromatography in calcium-containing buffer and as monomers of ∼75 kDa in dissociating buffer. The concentration of MASP-1 in donor sera (n = 105) was distributed log-normally with a median value of 11 µg/ml (range 4–30 µg/ml). Serum and citrate plasma levels were similar, while the values in ethylenediamine tetraacetic acid plasma were slightly lower and in heparin plasma were 1·5 times higher than in serum. MASP-1 was present at adult level at 1 year of age, while it was 60% at birth. In normal healthy individuals the level of MASP-1 was stable throughout a 2-month period. After induction of an acute-phase reaction by operation we found an initial short decrease, concomitant with an increase in C-reactive protein levels, followed by an increase, doubling the MASP-1 concentration after 2 days. The present data prepare the ground for studies on the associations of MASP-1 levels with disease.

Growing evidence suggests a prominent role of the complement system in the pathogenesis of cardio- and cerebrovascular diseases (CVD). Mannan-binding lectin-associated serine proteases (MASPs) MASP-1 and MASP-2 of the complement lectin pathway contribute to clot formation and may represent an important link between inflammation and thrombosis. MBL-associated protein MAp44 has shown cardioprotective effects in murine models. However, MAp44 has never been measured in patients with CVD and data on MASP levels in CVD are scarce. Our aim was to investigate for the first time plasma levels of MAp44 and MASP-1, -2, -3 concomitantly in patients with CVD. We performed a pilot study in 50 healthy volunteers, in stable coronary artery disease (CAD) patients with one-vessel (n = 51) or three-vessel disease (n = 53) and age-matched controls with normal coronary arteries (n = 53), 49 patients after myocardial infarction (MI) and 66 patients with acute ischaemic stroke. We measured MAp44 and MASP-1 levels by in-house time-resolved immunofluorometric assays. MASP-2 and MASP-3 levels were measured using commercial enzyme-linked immunosorbent assay kits. MASP-1 levels were highest in subacute MI patients and lowest in acute stroke patients. MASP-2 levels were lower in MI and stroke patients compared with controls and CAD patients. MASP-3 and MAp44 levels did not differ between groups. MASP or MAp44 levels were not associated with severity of disease. MASP and MAp44 levels were associated with cardiovascular risk factors including dyslipidaemia, obesity and hypertension. Our results suggest that MASP levels may be altered in vascular diseases. Larger studies are needed to confirm our results and elucidate the underlying mechanisms.

Primary immune deficiency (PID) disorders are clinically and molecularly heterogeneous diseases. T cell receptor excision circles (TRECs) and κ (kappa)-deleting excision circles (KRECs) are markers of T and B cell development, respectively. They are useful tools to assess T and B cell function and immune reconstitution and have been used for newborn screening for severe combined immunodeficiency disease (SCID) and agammaglobulinemia, respectively. Their profiles in several genetically confirmed PIDs are still lacking. The objective of this study was to determine TREC and KREC genomic profiling among various molecularly confirmed PIDs. We used real-time–quantitative polymerase chain reaction (RT–qPCR)-based triplex analysis of TRECs, KRECs and β-actin (ACTB) in whole blood genomic DNA isolated from 108 patients with molecularly confirmed PIDs. All agammaglobulinemia patients had low KREC counts. All SCIDs and Omenn syndrome patients secondary to mutations in RAG1, RAG2, DCLRE1C and NHEJ1 had low TREC and KREC counts. JAK3-deficient patients had normal KREC and the TREC count was influenced by the type of mutation. Early-onset ADA patients had low TREC and KREC counts. Four patients with zeta-chain-associated protein kinase 70 (ZAP70) had low TREC. All purine nucleoside phosphorylase (PNP) patients had low TREC. Combined immunodeficiency (CID) patients secondary to AK2, PTPRC, CD247, DCLREC1 and STAT1 had normal TREC and KREC counts. Most patients with ataxia–telangiectasia (AT) patients had low TREC and KREC, while most DOCK8-deficient patients had low TRECs only. Two of five patients with Wiskott–Aldrich syndrome (WAS) had low TREC counts as well as one patient each with bare lymphocyte syndrome (BLS) and chronic granulomatous disease. All patients with Griscelli disease, Chediak–Higashi syndrome, hyper-immunoglobulin (Ig)M syndrome and IFNGR2 had normal TREC and KREC counts. These data suggest that, in addition to classical SCID and agammaglobulinemia, TREC/KREC assay may identify ZAP70 patients and secondary target PIDs, including dedicator of cytokinesis 8 (DOCK8) deficiency, AT and some individuals with WAS and BLS.

The mechanisms of virus-induced airway hyperresponsiveness in asthma and allergy and the failure of host defence in patients suffering from secondary airway infections are still largely unknown. The aim of this study was to examine whether the presence of allergic rhinitis or susceptibility to recurrent sinusitis affects the structural and cellular changes in nasal mucosa during natural colds and convalescence. We compared the mucosal changes in biopsy samples during acute natural colds (days 2–4 of illness) and convalescence (3 weeks later) in patients with allergic rhinitis (n = 9), patients with susceptibility to sinusitis (n = 19) and healthy controls (n = 20). We saw similarly increased numbers of mucosal T and B lymphocytes and mast cells and increased vascular density during the acute colds compared to convalescence in all the three groups. The allergic subjects had elevated levels of eosinophils in the acute phase (P = 0·03), and the allergic and sinusitis-prone subjects had elevated levels of epithelial T cells (P = 0·04) and low levels of mast cells (P = 0·005) in convalescence compared to the control group. The sinusitis-prone subjects lacked intraepithelial cytotoxic cells in convalescence. In the allergic subjects, the reticular basement membrane was thicker in the acute phase compared to the convalescence (P = 0·05). These results suggest that various cells of the airways, including inflammatory and structural cells, are involved during viral respiratory infections in subjects with allergic rhinitis. The small numbers of mast cells and cytotoxic lymphocytes in the sinusitis-prone subjects may be related to their susceptibility to bacterial complications.