Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia.

BACKGROUND: Ataxia telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility and is caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the immunoglobulin deficiency, especially the variable severity, remain unelucidated. OBJECTIVE: We characterized the clinical impact of immunoglobulin deficiencies in AT and elucidated their mechanisms in AT. METHODS: We analyzed long-term immunoglobulin levels, immunophenotyping, and survival time in our cohort (n = 87, median age 16 years; maximum 64 years). Somatic hypermutation and class-switch junctions in B cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed, followed by RNA sequencing, to assess the effect of ATM inhibition. RESULTS: Only the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2 deficiencies did not. The immunoglobulin levels showed predominantly decreased IgG2 and IgA. Moreover, flow cytometric analysis demonstrated reduced naive B and T lymphocytes and a deficiency of class-switched IgG2 and IgA memory B cells. Somatic hypermutation frequencies were lowered in IgA- and IgG2-deficient patients, indicating hampered germinal center reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end joining during class-switch DNA repair. The in vitro class switching and proliferation were negatively affected by ATM inhibition. RNA sequencing analysis showed that ATM inhibitor influenced expression of germinal center reaction genes. CONCLUSION: Immunoglobulin deficiency in AT is caused by disturbed development of class-switched memory B cells. ATM deficiency affects both germinal center reaction and choice of DNA-repair pathway in class switching.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity.

Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.

Adverse reactions in a large cohort of patients with inborn errors of immunity receiving intravenous immunoglobulin.

BACKGROUND: Intravenous immunoglobulins (IVIg) are the major treatment in inborn errors of immunity (IEI) disorders; However, IVIg infusions show some adverse effects. We aimed to assess the adverse reactions of IVIg infusions. METHODS: Data of IVIg infusions in IEI patients were collected from 2011 to 2021. Totally, 363 IEI patients received IVIg regularly in Iran entered the study. The adverse reactions are classified regarding their severity and chronicity. RESULTS: 22,667 IVIg infusions were performed in the study. 157 patients (43.2%) and 1349 (5.9%) infusions were associated with at least one type of adverse reaction. The highest rates of adverse reactions were seen in severe combined immunodeficiency. Myalgia, chills, headache, fever, and hypotension were the most frequent adverse effects of IVIg. CONCLUSION: The reactions affect almost half of the patients mainly in the first infusions which necessitate the close observation of IEI patients receiving IVIg.

Altered NK-cell compartment and dysfunctional NKG2D/NKG2D-ligand axis in patients with ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a multisystem disorder caused by biallelic pathogenic variants in the gene encoding A-T mutated (ATM) kinase, a master regulator of the DNA damage response (DDR) pathway. Most A-T patients show cellular and/or humoral immunodeficiency that has been associated with cancer risk and reduced survival, but NK cells have not been thoroughly studied. Here we investigated NK cells of A-T patients with a special focus on the NKG2D receptor that triggers cytotoxicity upon engagement by its ligands (NKG2DLs) commonly induced via the DDR pathway on infected, transformed, and variously stressed cells. Using flow cytometry, we examined the phenotype and function of NK cells in 6 A-T patients as compared with healthy individuals. NKG2D expression was evaluated also by western blotting and RT-qPCR; plasma soluble NKG2DLs (sMICA, sMICB, sULBP1, ULBP2) were measured by ELISA. Results showed that A-T NK cells were skewed towards the CD56neg anergic phenotype and displayed decreased expression of NKG2D and perforin. NKG2D was reduced at the protein but not at the mRNA level and resulted in impaired NKG2D-mediated cytotoxicity in 4/6 A-T patients. Moreover, in A-T plasma we found 24-fold and 2-fold increase of sMICA and sULBP1, respectively, both inversely correlated with NKG2D expression. Overall, NK cells are disturbed in A-T patients showing reduced NKG2D expression, possibly caused by persistent engagement of its ligands, that may contribute to susceptibility to cancer and infections and represent novel targets for therapeutic interventions.

Simple Measurement of IgA Predicts Immunity and Mortality in Ataxia-Telangiectasia.

Patients with ataxia-telangiectasia (A-T) suffer from progressive cerebellar ataxia, immunodeficiency, respiratory failure, and cancer susceptibility. From a clinical point of view, A-T patients with IgA deficiency show more symptoms and may have a poorer prognosis. In this study, we analyzed mortality and immunity data of 659 A-T patients with regard to IgA deficiency collected from the European Society for Immunodeficiencies (ESID) registry and from 66 patients with classical A-T who attended at the Frankfurt Goethe-University between 2012 and 2018. We studied peripheral B- and T-cell subsets and T-cell repertoire of the Frankfurt cohort and survival rates of all A-T patients in the ESID registry. Patients with A-T have significant alterations in their lymphocyte phenotypes. All subsets (CD3, CD4, CD8, CD19, CD4/CD45RA, and CD8/CD45RA) were significantly diminished compared to standard values. Patients with IgA deficiency (n = 35) had significantly lower lymphocyte counts compared to A-T patients without IgA deficiency (n = 31) due to a further decrease of naïve CD4 T-cells, central memory CD4 cells, and regulatory T-cells. Although both patient groups showed affected TCR-ß repertoires compared to controls, no differences could be detected between patients with and without IgA deficiency. Overall survival of patients with IgA deficiency was significantly diminished. For the first time, our data show that patients with IgA deficiency have significantly lower lymphocyte counts and subsets, which are accompanied with reduced survival, compared to A-T patients without IgA deficiency. IgA, a simple surrogate marker, is indicating the poorest prognosis for classical A-T patients. Both non-interventional clinical trials were registered at clinicaltrials.gov 2012 (Susceptibility to infections in ataxia-telangiectasia; NCT02345135) and 2017 (Susceptibility to Infections, tumor risk and liver disease in patients with ataxia-telangiectasia; NCT03357978).

Accumulation of Cytoplasmic DNA Due to ATM Deficiency Activates the Microglial Viral Response System with Neurotoxic Consequences.

ATM (ataxia-telangiectasia mutated) is a PI3K-like kinase best known for its role in the DNA damage response (DDR), especially after double-strand breaks. Mutations in the ATM gene result in a condition known as ataxia-telangiectasia (A-T) that is characterized by cancer predisposition, radiosensitivity, neurodegeneration, sterility, and acquired immune deficiency. We show here that the innate immune system is not spared in A-T. ATM-deficient microglia adopt an active phenotype that includes the overproduction of proinflammatory cytokines that are toxic to cultured neurons and likely contribute to A-T neurodegeneration. Causatively, ATM dysfunction results in the accumulation of DNA in the cytoplasm of microglia as well as a variety of other cell types. In microglia, cytoplasmic DNA primes an antiviral response via the DNA sensor, STING (stimulator of interferon genes). The importance of this response pathway is supported by our finding that inhibition of STING blocks the overproduction of neurotoxic cytokines. Cytosolic DNA also activates the AIM2 (absent in melanoma 2) containing inflammasome and induces proteolytic processing of cytokine precursors such as pro-IL-1ß. Our study furthers our understanding of neurodegeneration in A-T and highlights the role of cytosolic DNA in the innate immune response.SIGNIFICANCE STATEMENT Conventionally, the immune deficiencies found in ataxia-telangiectasia (A-T) patients are viewed as defects of the B and T cells of the acquired immune system. In this study, we demonstrate the microglia of the innate immune system are also affected and uncover the mechanism by which this occurs. Loss of ATM (ataxia-telangiectasia mutated) activity leads to a slowing of DNA repair and an accumulation of cytoplasmic fragments of genomic DNA. This ectopic DNA induces the antivirus response, which triggers the production of neurotoxic cytokines. This expands our understanding of the neurodegeneration found in A-T and offers potentially new therapeutic options.

Immune competence and respiratory symptoms in patients with ataxia telangiectasia: A prospective follow-up study.

Ataxia telangiectasia is a multi-system disorder characterized by progressive cerebellar ataxia, malignancies, chronic pulmonary disease and immunodeficiency. The aim of our study was to determine the immune competence and prevalence of respiratory infections and/or chronic cough in classical A-T patients compared to age-matched healthy controls. STUDY DESIGN: We recruited 20 classical A-T not treated by immunoglobulins and 21 healthy age-matched control patients. The caregivers were advised to keep a daily diary with the following items (daytime and nighttime cough, runny nose, fever), number of cold episodes, number of antibiotic treatments. RESULTS: Patients with A-T showed significant differences compared to healthy controls in symptom score, daytime and nighttime cough, days with symptoms and missed days in kindergarten/school. Severe infections with hospitalization occurred rarely. Respiratory symptoms did not correlate with immunoglobulin levels in A-T patients. CONCLUSIONS: Mild symptoms like chronic cough were present in A-T patients, possibly indicating ongoing silent crippling disease.

A novel ATM mutation associated with elevated atypical lymphocyte populations, hyper-IgM, and cutaneous granulomas.

Ataxia-Telangiectasia (AT) is an immunodeficiency most often associated with T cell abnormalities. We describe a patient with a hyper-IgM phenotype and immune cell abnormalities that suggest a distinct clinical phenotype. Significant B cell abnormalities with increased unswitched memory B cells, decreased naive transitional B cells, and an elevated frequency of CD19+CD38loCD27-CD10-CD21-/low B cells expressing high levels of T-bet and Fas were demonstrated. The B cells were hyporesponsive to in vitro stimulation through the B cell receptor, Toll like receptors (TLR) 7 and 9, and CD40. T cell homeostasis was also disturbed with a significant increase in γδ T cells, circulating T follicular helper cells (Tfh), and decreased numbers of T regulatory cells. The ATM mutations in this patient are posited to have resulted in the perturbations in the frequencies and distributions of B and T cell subsets, resulting in the phenotype in this patient. KEY MESSAGES: A novel mutation creating a premature stop codon and a nonsense mutation in the ATM gene are postulated to have resulted in the unique clinical picture characterized by abnormal B and T cell populations, lymphocyte subset dysfunction, granuloma formation, and a hyper-IgM phenotype. CAPSULE SUMMARY: A patient presented with ataxia-telangiectasia, cutaneous granulomas, and a hyper-IgM phenotype; a novel combination of mutations in the ATM gene was associated with abnormal distributions, frequencies, and function of T and B lymphocyte subsets.

Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency.

BACKGROUND: Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). OBJECTIVE: We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. METHODS: Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. RESULTS: Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. CONCLUSIONS: Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature.

Ataxia Telangiectasia and Cancer Predisposition: Challenges in Management.

Immune dysregulation and predisposition to malignancies are critical comorbidities in children affected with ataxia telangiectasia. In addition, these children exhibit increased toxicity to conventional cancer therapy and dose reductions have been proposed to prevent life threatening adverse effects. These modifications to the treatment regimen may result in suboptimal outcomes for these patients. Our report of 3 children with ataxia telangiectasia and cancer highlight the immense challenges in the management of these children, underlining the need for the development of novel, biological agents with reduced acute and long-term side effects in the treatment of cancers in these children.

Elevated IgM levels as a marker for a unique phenotype in patients with Ataxia telangiectasia.

BACKGROUND: Ataxia telangiectasia (AT) is a rare, multi-systemic, genetic disorder. Mutations in the ATM gene cause dysfunction in cell-cycle, apoptosis and V (D) J recombination leading to neurodegeneration, cellular, humoral immunodeficiencies and predisposition to malignancies. Previous studies have suggested that a sub-group of AT patients with elevated IgM levels have a distinct and more severe phenotype. In the current study we aimed to better characterize this group of patients. METHODS: We performed a retrospective review of 46 patient records, followed from January 1986 to January 2015 at the Israeli National AT Center. Demographic, clinical, radiological, laboratory data was reviewed and compared between AT patients with elevated IgM levels (EIgM) and patients with normal IgM levels (NIgM). RESULTS: 15/46(32.6%) patients had significantly elevated IgM levels. This group had a unique phenotype characterized mainly by increased risk of infection and early mortality. Colonization of lower respiratory tract with Mycobacterium gordonae and Pseudomonas aeruginosa as well as viral skin infections were more frequent in EIgM patients. Patients with NIgM had a significantly longer survival as compared to patients with EIgM but had an increased incidence of fatty liver or cirrhosis. T-cell recombination excision circles and kappa-deleting element recombination circle levels were significantly lower in the EIgM group, suggesting an abnormal class switching in this group. CONCLUSIONS: EIgM in AT patients are indicative of a more severe phenotype that probably results from a specific immune dysfunction. EIgM in AT should be considered a unique AT phenotype that may require different management.

B-cell subsets imbalance and reduced expression of CD40 in ataxia-telangiectasia patients.

BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-γ of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity.

Ataxia-telangiectasia: Immunodeficiency and survival.

Ataxia-telangiectasia (AT) is a neurodegenerative disorder characterized by ataxia, telangiectasia, and immunodeficiency. An increased risk of malignancies and respiratory diseases dramatically reduce life expectancy. To better counsel families, develop individual follow-up programs, and select patients for therapeutic trials, more knowledge is needed on factors influencing survival. This retrospective cohort study of 61 AT patients shows that classical AT patients had a shorter survival than variant patients (HR 5.9, 95%CI 2.0-17.7), especially once a malignancy was diagnosed (HR 2.5, 95%CI 1.1-5.5, compared to classical AT patients without malignancy). Patients with the hyper IgM phenotype with hypogammaglobulinemia (AT-HIGM) and patients with an IgG2 deficiency showed decreased survival compared to patients with normal IgG (HR 9.2, 95%CI 3.2-26.5) and patients with normal IgG2 levels (HR 7.8, 95%CI 1.7-36.2), respectively. If high risk treatment trials will become available for AT, those patients with factors indicating the poorest prognosis might be considered for inclusion first.

Noninfectious Granulomas: A Sign of an Underlying Immunodeficiency?

Primary immunodeficiency disorders, such as ataxia-telangiectasia (A-T), may rarely be associated with cutaneous granulomas without an identifiable infection. The authors report a case of a 3-year-old boy with A-T who presented with two persistent ulcerated erythematous nodules. Histopathology was consistent with a granulomatous process secondary to A-T, without an infectious origin. Partial improvement was noted with clobetasol propionate 0.05% cream applied twice daily under occlusion. Of note, the presence of multiple noninfectious granulomas in a child may be the initial sign of an immune deficiency and should alert the astute clinician to investigate for an underlying primary immunodeficiency. Herein, the authors discuss the associations of noninfectious granulomas and primary immunodeficiency disorders and present management options for these difficult-to-treat lesions.

Comparison of major lymphocyte subpopulations and recent thymic emigrants in patients with ataxia telangiectasia and age-matched healthy groups.

BACKGROUND: Ataxia telangiectasia (A-T) is a genetic disorder caused by the homozygous mutation of the A-T mutated gene. It is frequently associated with variable degrees of cellular and humoral immunodeficiency. However, the immune defects in A-T patients are not well characterized. To the best of our knowledge, no studies have focused on the major lymphocyte subpopulations and recent thymic emigrants of A-T patients in comparison with age-matched healthy controls. METHODS: Following the European Society for Immunodeficiencies criteria, 17 patients diagnosed with A-The, and 12 age-matched healthy children were assigned to the study. Both patients and healthy controls were grouped as 1-5, 6-10, 11-15, and 15+ years. By using a flow cytometer, major lymphocyte subpopulations and CD4+CD45RA+CD31+ recent thymic emigrants were determined as percentage and absolute cell numbers and compared. RESULTS: No significant differences in all lymphocyte subpopulations were observed between the age groups of A-T patients. Compared to the healthy controls, there was a decrease in T cells, effector memory T4 cells, B cells, naïve B cells, naïve T4 cells, switched B cells, and recent thymic emigrants and an increase in active T8 cells and non-switched B cells in the percentage and absolute number of some cell populations in the A-T group. CONCLUSIONS: This study showed that effector functions in some cell lymphocyte populations were decreased in A-T patients.

Class switch recombination process in ataxia telangiectasia patients with elevated serum levels of IgM.

Ataxia telangiectasia (AT) is a rare primary immunodeficiency disorder with various clinical manifestations. Increased serum levels of IgM and recurrent infections, mainly sinopulmonary infections, can be the presenting feature in a number of AT patients and may be initially misdiagnosed as hyper-IgM (HIgM) syndrome. This study was designed to investigate class switch recombination (CSR) as a critical mechanism in B lymphocytes' maturation to produce different isotypes of antibody in response to antigen stimulation in AT cases with HIgM presentation. Quantitative IgE production after stimulation by IL-4 and CD40L was considered as an indicator for CSR function. We also compared their results with sex and age matched AT patients without HIgM presentation. We report four AT patients with recurrent infections during infancy and high serum levels of IgM. Laboratory evaluations revealed defective CSR while none of the three AT patients without HIgM presentation had a defect in the CSR process. The characterized defect in AT is a mutation in the ataxia telangiectasia mutated (ATM) gene. This gene may result in CSR defects due to impaired DNA break repair. A special association between AT and HIgM may indicate a new subgroup of AT patients according to their clinical phenotype and CSR condition.

Disturbed B and T cell homeostasis and neogenesis in patients with ataxia telangiectasia.

OBJECTIVE: Ataxia telangiectasia (AT) is a rare genetic, multi-system disorder characterized by neurodegeneration, chromosome instability, B and T cell immunodeficiency and a predisposition to cancer. We examined immunologic parameters reflecting cell development and proliferation and their relevancy to the clinical phenotype in affected individuals. PATIENTS AND METHODS: AT patients from the AT National Clinic in Israel underwent immunological investigation. Their T and B cell workup included lymphocyte subset counts, immunoglobulin levels, responses to mitogenic stimulations, TCR-Vß families and BCR immunoglobulin heavy chain spectratyping, TCR rearrangement excision circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). RESULTS: Thirty-seven AT patients (median age 12.7 years, range 4.2-25.1) were evaluated. CD20 B and CD3 T lymphocytes were decreased in 67 % and 64 % of the patients, respectively, while only 33 % of the patients had reduced lymphoproliferative responses. Almost all AT patients displayed extremely low TRECs and KRECs levels, irrespective of their age. Those levels were correlated to one another and to the amounts of CD3+ and CD20+ cells, respectively. Abnormal TCR-Vß repertoires were found with different degrees of clonality or reduced expression in these AT patients. There was no clear clustering of expansions to specific TCR-Vß genes. PCR spectratyping analysis of the FR2 IgH BCR gene rearrangements in peripheral blood was abnormal in 50 % of the patients. CONCLUSION: The immunodeficiency associated with AT is combined, remains low over time and not progressive. It is characterized by low TREC and KREC copies suggestive of abnormal T and B cell neogenesis.

Immune deficiency in Ataxia-Telangiectasia: a longitudinal study of 44 patients.

Ataxia-Telangiectasia (A-T) is a genetic condition leading to neurological defects and immune deficiency. The nature of the immune deficiency is highly variable, and in some cases causes significant morbidity and mortality due to recurrent sinopulmonary infections. Although the neurological defects in A-T are progressive, the natural history of the immune deficiency in A-T has not been evaluated formally. In this study we analyse the clinical history and immunological data in 44 patients with A-T who attended the National Ataxia-Telangiectasia clinic in Nottingham between 2001 and 2011. Using patient medical records and Nottingham University Hospitals (NUH) National Health Service Trust medical IT systems, data regarding clinical history, use of immunoglobulin replacement therapy, total immunoglobulin levels, specific antibody levels and lymphocyte subset counts were obtained. T cell receptor spectratyping results in some patients were already available and, where possible, repeat blood samples were collected for analysis. This study shows that subtle quantitative changes in certain immunological parameters such as lymphocyte subset counts may occur in patients with A-T over time. However, in general, for the majority of patients the severity of immune deficiency (both clinically and in terms of immunological blood markers) does not seem to deteriorate significantly with time. This finding serves to inform the long-term management of this cohort of patients because, if recurrent respiratory tract infections present later in life, then other contributory factors (e.g. cough/swallowing difficulties, underlying lung disease) should be investigated aggressively. Our findings also offer some form of reassurance for parents of children with A-T, which is otherwise a progressively severely debilitating condition.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

OBJECTIVE: To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). MATERIALS: We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. TREATMENT: The cells were cultured with or without glutamine or GSH. METHODS: ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. RESULTS: While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. CONCLUSION: Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.