Contribution of ADAM33 polymorphisms to the population risk of asthma.

BACKGROUND: ADAM 33 is the first gene identified as a candidate for asthma by positional cloning techniques, with association studies reaching impressive statistical significance. It has a postulated role in myogenesis, airway modelling, and signalling via protein shedding. Concerns over the methodology of the initial study have led to several attempts at replication, with inconsistent results. METHOD: To clarify the role of ADAM33 in determining the risk of asthma in the general population, new transmission disequilibrium and case-control studies were undertaken followed by a meta-analysis of all existing data. RESULTS: Studies in Icelandic and UK populations revealed no association when taken in isolation. The meta-analysis, however, showed that the F+1 and ST+7 variants were significantly associated with asthma in both types of study. CONCLUSIONS: The additional risk imparted by this variation would account for 50,000 excess asthma cases in the UK alone. This study also demonstrates the size of study required to investigate such hypotheses adequately.

Association between IL-1beta/TNF-alpha-induced glucocorticoid-sensitive changes in multiple gene expression and altered responsiveness in airway smooth muscle.

The pleiotropic cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha have been implicated in the pathophysiology of asthma. To elucidate the role of these cytokines in the pro-asthmatic state, the effects of IL-1beta and TNF-alpha on airway smooth muscle (ASM) responsiveness and ASM expression of multiple genes, assessed by high-density oligonucleotide array analysis, were examined in the absence and presence of the glucocorticoid dexamethasone (DEX). Administration of IL-1beta/TNF-alpha increased ASM contractility to acetylcholine and impaired ASM relaxation to isoproterenol. These pro-asthmatic- like changes in ASM responsiveness were associated with IL-1beta/ TNF-alpha-induced mRNA expression of a host of proinflammatory genes that regulate transcription, cytokines and chemokines, cellular adhesion molecules, and various signal transduction molecules that regulate ASM responsiveness. In the presence of DEX, the changes induced in ASM responsiveness were abrogated, and most of the IL-1beta/TNF-alpha-mediated changes in proinflammatory gene expression were repressed, although mRNA expression of a small number of genes was enhanced by DEX. Collectively, the observations support the concept that, together with its role as a regulator of airway tone, in response to IL-1beta/TNF-alpha, the ASM expresses a host of glucocorticoid-sensitive genes that contribute to the altered structure and function of the airways in the pro-asthmatic state. We speculate that glucocorticoid-sensitive, cytokine-induced pathways involved in ASM cell signaling represent important targets for new therapeutic interventions.

Allelic frequencies and patterns of single-nucleotide polymorphisms in candidate genes for asthma and atopy in Iceland.

Numerous asthma and atopy loci have been reported in studies demonstrating associations of the asthma-related phenotypes atopy, elevated IgE levels, and bronchial hyperresponsiveness with alleles of microsatellite markers and single-nucleotide polymorphisms (SNPs) within specific cytokine/chemokine and IgE-regulating genes. Although the studies reporting these observations are compelling, most of them lack statistical power. We assessed the nature, pattern, and frequency of SNPs in 24 candidate genes in Iceland and looked for associations with asthma and atopy. We identified 42 SNPs with an average minor allele frequency of 20.3% (asthma) and 20.7% (control). Twenty SNPs (48%) were within coding sequences and 90% of those led to a predicted change in protein sequence. No differences were detected in the allelic frequencies of SNPs in any of these candidate genes between control subjects and the patients with atopic asthma. Moreover, linkage analysis that included 269 patients with atopic asthma uncovered no evidence of linkage to markers associated with these genes. We conclude that this study has failed to produce evidence in support of the notion that variations within these 24 candidate atopy and asthma genes significantly influence the expression of the atopic asthmatic phenotype or contribute to the susceptibility of atopic asthma.

Kinetics of the T-cell receptor CD4 and CD8 V beta repertoire in HIV-1 vertically infected infants early treated with HAART.

OBJECTIVES: To determine the kinetics and the relationship between the T-cell receptor V beta (TCRBV) complementary determining region 3 length, the CD4 T-cell count and HIV viral load changes in HIV-1 infected infants treated early with highly active antiretroviral therapy (HAART) during 1 year of follow-up. DESIGN: Two HIV-1 vertically infected infants, two HIV-1 vertically exposed uninfected and two healthy controls were analysed by spectratyping. Evaluation of viral load, CD4 naive and memory cell counts and a proliferation test were also carried out. METHODS: Twenty-six families and subfamilies of the TCR on CD4 and CD8 T cells were analyzed by spectratyping. Flow cytometric analysis on peripheral blood mononuclear cells for CD4CD45Ra, CD4CD45Ro, CD8CD38, proliferation tests and plasma viral load measurements were performed at baseline, 1, 6 and after 12 months of therapy. RESULTS: HAART induced a marked reduction of viral load in both HIV-1 infected infants and an increase to normal CD4 T-cell count in the symptomatic infant. At baseline the TCRBV family distribution in the majority of CD8 and a few of the CD4 T cells was highly perturbed, with several TCRBV families showing a monoclonal/oligoclonal distribution. During HAART a normalization of the TCR repertoire in both CD8 and CD4 subsets occurred. TCR repertoire normalization was associated with a good virological and immunological response. CONCLUSION: These results suggest that complete and early virus replication control as a result of early HAART leads to a marked reduction of T-cell oligoclonality and is an essential prerequisite to the development of a polyclonal immune response in HIV-1 infected infants.

Diverse T-cell receptor CDR3 length patterns in human CD4+ and CD8+ T lymphocytes from newborns and adults.

T cells are essential in the initiation and maintenance of immune responses. Specific interaction between T cells and a presumptive antigen occurs through recognition of an MHC-peptide complex by the T-cell receptor (TCR). The complementarity-determining region (CDR) 3 of the TCR has direct contact with the peptide. Here we describe CDR3 length variability of six different TCRBV gene families of CD4+ and CD8+ umbilical cord (UC) and peripheral blood (PB) T cells. Amplified products spanning the TCR CDR3 regions from CD4+ PB, CD4+ UC and CD8+ UC blood T cells typically displayed Gaussian-like distributions. In contrast, profound and frequent perturbations were recorded in CD8+ PB lymphocytes, with a non-Gaussian pattern in more than half of the samples studied. A substantial portion of the perturbed CD8+ subsets were clonal or oligoclonal, as determined by CDR3-length restriction, TCRBJ gene usage and nucleotide sequencing. This implies that the conditions for shaping and maintenance of the peripheral TCR repertoire are profoundly different for CD8+ and CD4+ T cells.

T-cell expansions with conserved T-cell receptor beta chain motifs in the peripheral blood of HLA-DRB1*0401 positive patients with necrotizing vasculitis.

T lymphocytes are implicated in the pathogenesis of systemic vasculitis such as Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN). In the present study, we have characterized in detail the T-cell receptor (TCR) of peripheral blood T cells from eight vasculitis patients of known HLA class II genotypes. We used flow cytometry to outline the exact TCR V gene expression, complementarity determining region 3 (CDR3) fragment analysis to estimate the degree of clonality and cDNA sequencing to define the exact TCR or beta chain sequences. The TCR CDR3 region interacts with antigenic peptides presented by HLA molecules, and it is normally immensely diverse. It was therefore of particular interest to identify a common dominating TCR BV8-F/L-G-G-A/Q-G-J2S3 beta chain sequence in the CD4(+) T cells of four unrelated vasculitis patients. Furthermore, this BV8-associated CDR3 motif was linked to the HLA-DRB1*0401 allele, as well as to active disease and/or an established BV8(+) CD4(+) T-cell expansion. In contrast, age- and HLA-matched patients with rheumatoid arthritis did not harbor the described BV8 motif. These results strongly suggest that BV8(+) CD4(+) T cells with the described CDR3 motif recognize a specific antigen presented by DR4 molecules, indicating the existence of a common vasculitis-associated antigen.

Oligoclonal T cells in human cancer.

Many solid tumors are characterised by the infiltration of lymphocytes and their presence has been correlated with a more favourable prognosis. These tumor-infiltrating lymphocytes (TIL), have been shown to possess specific cytolytic reactivity towards autologous tumours, thus suggesting that tumour cells may express antigens capable of eliciting an immune response. Expression of such tumour-associated antigens (TAA) in combination with appropriate accessory signals would lead to the in vivo accumulation of T cells with anti-tumour specificity. Analysis of the composition of the specific T-cell receptor (TCR) of TIL could thus provide information on the nature of the antigen(s) recognised by TIL. In this review, different aspects of the presence of clonal T cells in patients with cancer are discussed.

Correlation between HIV sequence evolution, specific immune response and clinical outcome in vertically infected infants.

OBJECTIVE: To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1. DESIGN: Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3). METHODS: Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time. RESULTS: Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of children's sera to react with synthetic peptides representing the autologous virus sequence was determined. CONCLUSION: Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

The role of virologic and immunologic factors in mother-to-child transmission of HIV-1.

PROBLEM: More than 90% of human immunodeficiency virus type 1 (HIV-1) infection in children is acquired by mother-to-child transmission. However, infection of the child occurs in between 14 and 35% of cases. METHOD OF STUDY: To understand the mechanisms involved in HIV-1 transmission, we have investigated the antigenic, molecular, and phenotypic characteristics of the virus harbored in infected mothers and their children. RESULTS: A clear correlation was observed between the transmission of the virus and the isolation of viral variants with a rapidly replicating and syncytium-inducing phenotype from the mother. Furthermore, non-transmitting mothers were able to neutralize several primary isolates more frequently than transmitting mothers. The comparison of the viral phenotype and genotype of mother-child pairs showed that the transmitted virus did not have common features, suggesting that transmission is usually not a selective process. CONCLUSIONS: This study suggests that transmission is governed by an interaction of both viral and immunological factors. The results obtained indicate that different strategies can be applied for the prevention of transmission.

T cell repertoire in patients with multiple myeloma and monoclonal gammopathy of undetermined significance: clonal CD8+ T cell expansions are found preferentially in patients with a low tumor burden.

The T cell receptor (TCR) variable (V) gene repertoire was analyzed in patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 17), multiple myeloma (MM) stage I (n = 16), MM stages II/III (n = 31) and age-matched controls (n = 27) by immunofluorescence and flow cytometry using a panel of mouse monoclonal antibodies (mAb) (n = 10) against TCR V alpha and V beta gene products. T cell expansion was defined as a value > or = thrice the normal median value for each respective TCR V mAb. Fifty-three percent of all patients displayed CD8+ expansion(s) as compared to 30% of age-matched controls (p < 0.001). Within the CD4 subset, 18% of the patients displayed T cell expansion(s) in comparison to 11% of the controls (not significant). Interestingly, the CD8+ expansion(s) were more frequently noted in patients with a low tumor burden (MGUS/MMI) (73%) as compared to those with advanced disease (MM II/III) (32% and control donors (30%) (p < 0.01). Likewise, multiple CD8+ expansions (two or more) were more common in MGUS/MM I patients than in MM II/III and controls (p < 0.01). The T cell expansions were stable over time in patients with a stable disease. A high degree of clonality of the expansions was detected by TCR CDR3 fragment length analysis, determination of J beta gene usage and nucleotide sequencing. The frequent finding of oligoclonal CD8+ T cell expansions in patients with a low tumor mass, but not in patients with advanced disease justifies further work in order to identify the relevance of expanded CD8+ T cells. In one patient with T cell reactivity against the autologous myeloma idiotype, two expansions within the CD8 population (V beta 3 and V beta 5.2 respectively) displayed no reactivity against the idiotype. Instead, idiotype recognition was confined to a CD8 non-expanded V beta 22+ T cell population, with a highly restricted TCR usage (CDR3 fragment length analysis).

Role of immunity in maternal-infant HIV-1 transmission.

Factors influencing human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission include both immunological and virological parameters: higher viral loads have been associated with clinical stage of HIV-1-infected individuals as well as higher risk of mother-to-child transmission. Furthermore, we have shown that transmitting mothers more frequently harbour HIV-1 isolates with rapid/high syncytium-inducing (SI) biological phenotype than non-transmitting mothers do. Genetically homogeneous virus populations have been found in HIV-1-infected children at birth, in contrast to the heterogeneous virus populations often found in their infected mothers. This observation suggests that a few virus variants are transmitted or initially are replicating in the child. By comparing the HIV-1 gp120 V3 region of sequentially obtained samples from infected children with samples obtained from their mothers at delivery we found, however, that multiple variants of HIV-1 with different outgrowth kinetics can be transmitted. In addition, we have obtained results indicating an impaired ability of the immune response to adapt to the sequence evolution of HIV-1 in transmitting mothers, as assessed by measuring serum reactivities to peptides representing selected yet closely related V3 sequences. By analysing the presence of antibodies in maternal serum at delivery, which neutralize autologous isolates as well as other primary virus isolates, we have indications that a protective immunity in HIV-1 mother-to-child transmission might exist. Immunotherapy has been assessed in infected adult individuals by passive immunization with a variety of HIV-1-specific antibody products. Data from these studies indicated a differential response to therapy according to the stage of the disease. Active vaccine strategies, including envelope glycoproteins, pursued so far in seronegative adult subjects have shown limitations because broadly neutralizing antibodies, such as can be found in infected individuals, have not been evoked. Further investigations are therefore needed to give support for the potential use of either passive and/or active immunization for the prevention of HIV-1 mother-to-child transmission.

RT-PCR topography of chronic psoriasis skin based on analysis of T-cell receptor B variable region gene usage.

Psoriasis is a hyperproliferative inflammatory disease and 70% of patients develop a chronic plaque form. The pathogenesis of psoriasis is not known but evidence exists that T cells play a crucial role. The T cell V-gene receptor repertoire from psoriasis skin (different layers) was compared with peripheral blood T cells by employing RNA polymerase chain reaction (PCR) amplification. T cell receptor (TCR) BV 5.1, 11, 12, 13.1 and 16 were utilized to a significantly higher degree in areas close to the basal layers when compared to CD4+, CD8+ or unfractionated blood T cells from the same patients, whereas only BV11 and 13.1 genes of T cells from deeper layers of the dermis showed such a skewed usage. No biased usage of TCRBV genes was observed in superficial layers or in whole skin. Furthermore, T cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin psoriatic T cells to be poly- or oligoclonal. In conclusion, we show that TCRBV gene usage from different layers of psoriatic skin has a different pattern compared with the corresponding gene usage in circulating peripheral blood T cells. This pattern may implicate possible skin-associated antigen or superantigens activating a limited number of T cells in areas of skin close to basal layers, which in turn could promote keratinocyte proliferation.

Mechanisms of escape from CD8+ T-cell clones specific for the HER-2/neu proto-oncogene expressed in ovarian carcinomas: related and unrelated to decreased MHC class 1 expression.

We have developed an in vitro model to study mechanisms by which ovarian tumor cells that over-express the HER-2/neu proto-oncogene escape recognition by TCD8+. Nine tumor-specific, HLA A2-restricted TCD8+ clones were isolated from 2 ovarian tumor-specific TCD8+ lines derived from tumor-infiltrating or -associated lymphocytes. Of these, 2 clones recognized the previously defined HER-2/neu epitope E75 (a.a. 369-377) and one recognized the C85 epitope (a.a. 971-979), whereas the specificity of the remaining 6 clones was unknown. Three different tumor escape variants (EVC8, EVC22 and EVC36) were produced by co-culturing an ovarian tumor line over-expressing HER-2/neu with these autologous TCD8+ clones. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER-2/neu, ICAM-1 or LFA-3 molecules was found. There was a correlation between the level of tumor-specific recognition and HLA A2 expression among the tumor clones isolated from 2 of the escape variants (EVC8 and EVC36). In contrast, high HLA A2-expressing tumor clones isolated from the EVC22 variant, or EVC22 which had regained high HLA A2 expression through IFN-gamma treatment, were not recognized by the HER-2/neu-specific TCD8+ clone C-22. No mutations were found in the cDNA or the genomic DNA derived from the PCR product corresponding to a 496 bp fragment including the region coding for the E75 epitope of the HER2/neu gene in the EVC22 variant. Collectively, this in vitro model underlines the importance of decreased expression of the HLA restriction element for escape from tumor-specific TCD8+ but also demonstrates that additional mechanisms exist.

Clonal CD8+ and CD52- T cells are induced in responding B cell lymphoma patients treated with Campath-1H (anti-CD52).

Five patients with non-Hodgkin's lymphoma (NHL) and 4 patients with chronic lymphocytic leukaemia (CLL) were treated with the CDR-grafted (rat x human) monoclonal antibody (mAb) Campath-1H (anti-CD52). Tumour regression was noted preferentially in peripheral blood and in the bone marrow but lymph nodes were less affected. Normal blood B and T cells were profoundly reduced in all patients whereas CD16+ NK cells and CD14+ monocytes decreased marginally. In all responding CLL patients CD52-negative T but not B cells appeared during treatment and persisted for several months (4-19+) during unmaintained follow-up. Clonal T cells defined as a predominance of a single T cell receptor (TCR) V gene usage, in one case verified by TCR CDR3 fragment analysis and nucleotide sequencing, emerged within the CD52-/CD8+ cell population during Campath-1H therapy in 2 CLL patients, both achieving a long-lasting remission. The increase in CD8+ T cell expansions (up to 23-fold) during unmaintained remission and follow-up suggest that the clonal CD8+ cells may represent regulatory T cells controlling the growth of the tumour B cell clone. Clonal T cells might thus be a target for an immune therapeutic intervention in B cell tumours.

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

OBJECTIVE: To investigate the T-cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV-1 in order to discern possible perturbations in TCR usage as a consequence of HIV-1 infection. DESIGN: Blood samples from five HIV-1-infected and six non-infected children born to HIV-1-seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. METHODS: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V alpha and V beta gene products and antibodies to CD4 and CD8 was performed. RESULTS: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V alpha and V beta gene products was seen in HIV-1-infected children (four out of five) but was rarely detected in uninfected children. CONCLUSION: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V alpha/V beta gene products in some HIV-1 vertically infected infants similar to those observed during primary infection in adults.

RT-PCR based analysis of T-cell receptor B variable region gene usage in normal human breast skin resident T lymphocytes (SRT).

The skin interfaces directly with the external environment that contains innumerable infectious agents. Therefore, an appropriate and rapid immunologic response is required to preserve internal homeostasis. An essential feature of the "skin immuno system' (SIS) is the presence of substantial numbers of T cells in normal skin. The T-cell receptor repertoire from normal human breast skin was analysed quantitatively and qualitatively by using PCR amplification of reverse transcribed RNA, T-cell receptor BV3 and BV14 gene usage was increased in skir T lymphocytes in all individuals tested (n = 8) compared to peripheral blood CD4+ and CD8+ T lymphocytes from the same individuals. The T-cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin T-cell BV3 and BV14 gene usage to be predominantly polyclonal. Superantigen stimulation of T cells in human skin is considered a likely explanation of the present finding.

Increased frequency of abnormal gamma delta T cells in blood of patients with inflammatory bowel diseases.

We have previously reported a preferential usage of V delta 1/V gamma 8 on TCR-gamma-delta-bearing intraepithelial lymphocytes of the normal human intestine as well as in the inflamed synovial tissue of patients with rheumatoid arthritis. The aim of the present study was to analyze V gene segment usage by gamma delta T cells of the intestine and peripheral blood (PB) from patients with inflammatory bowel disease. Freshly isolated lymphocytes were analyzed by flow cytometry using a panel of V gene subset-specific mAbs. The relative proportion of PB TCR-gamma delta+ cells was increased in patients with Crohn's disease as compared with controls. Interestingly, an increased proportion of PB gamma delta T cells of patients with ulcerative colitis or CrD expressed V delta and V gamma genes typically used by intraepithelial lymphocytes. Thus, increased proportions of V delta 1+ and V gamma 8+ cells were found in the PB of both patient groups. The majority of TCR-gamma delta+ intraepithelial lymphocytes (IEL) in inflamed and noninflamed intestine expressed V delta 1/V gamma 8, and a substantial proportion of V gamma(2,3,4)+ cells were found. These observations suggest a disease-associated appearance of "gut-like" gamma delta T cells in the periphery. Moreover, two patients had a large proportion of gamma delta T cells in their PB of which the majority expressed two distinct V gamma proteins. Both of these patients had a short duration of disease (1 and 10 mo, respectively) and the relative proportion of gamma delta T cells decreased in concert with stabilization of disease. Interestingly, one of the patients had a clonal expansion of a V gamma dual-expressing gamma delta T cell in the PB.

Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma.

Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-alpha and IFN-gamma mRNA were expressed non-selectively in tumors, PBMC and normal rental tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-1 alpha, IL-6, TNF-alpha and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.