Exploring Autoimmunity in a Cohort of Children with Genetically Confirmed Aicardi–Goutières Syndrome

Purpose

The purpose of this study was to explore the presence of autoimmune manifestations and characterize the autoantibody production in a cohort of patients with Aicardi–Goutières syndrome (AGS).

Methods

Seventeen patients with a genetically-confirmed diagnosis of AGS were recruited. At the time of enrollment, past medical and family history was reviewed, looking for possible signs or symptoms of autoimmune disorders. Blood samples were taken, for the detection of a panel of autoantibodies: anti-nuclear, anti-double-stranded-DNA, anti-nucleosome, anti-extractable nuclear antigens, anti-cardiolipin IgG/IgM, anti-β2glycoprotein I IgG/IgM, and anti-neutrophil cytoplasmic. We also measured complement levels determined as C3 and C4 quantification and total complement activity, measured as CH50.

Results

Nine of seventeen patients presented with at least one first- or second-degree relative with a history of autoimmune diseases (the childrens’ mother or grand-mother in the majority of cases). A specific autoimmune disease was present in only one AGS patient, namely an autoimmune thyroiditis. Autoantibodies were present in 9/17 patients, with different patterns of positivity. Complement levels were normal in all the patients. There was no correlation between auto-antibody production and personal or family history of autoimmune diseases.

Conclusions

Definite autoimmune diseases are not common in patients with AGS. Autoantibodies are mainly directed towards nucleic acids-containing elements but seem not to be pathogenic and, rather, may represent an epiphenomenon of the enhanced interferon production.

     

Detection of Campylobacter jejuni by Culture and Real-Time PCR in a French Cohort of Patients with Guillain-Barré Syndrome

Bacteriological culture and real-time PCR (RT-PCR) were used to detect Campylobacter jejuni in fecal samples from a French cohort of 237 patients with Guillain-Barré syndrome (GBS). We provide evidence that diverse serotypes and genotypes of C. jejuni are a major trigger of GBS in France.Guillain-Barré syndrome (GBS) is a postinfectious neurological disease, with a median estimated annual incidence of 1.3/100,000 in developed countries (7). Campylobacter jejuni is the most frequent triggering agent, accounting for 15 to 40% of GBS cases in North America, Australia, and Europe (6, 14, 16, 20). The majority of cases of C. jejuni-associated GBS in Japan involve serotype O:19 (18), and the majority of cases in South Africa involve serotype O:41 (12). A much greater diversity of serotypes (4, 17) and genotypes (4) has been reported in Europe. The pathogenesis of C. jejuni-associated GBS has been linked to antiganglioside autoantibodies generated as a result of the molecular mimicry displayed by ganglioside-like oligosaccharides present in the lipopolysaccharide of certain C. jejuni strains (21). More recently, it has also been linked to the presence of particular classes of the lipo-oligosaccharide (LOS) biosynthesis locus, with most GBS strains having class A or B loci (10, 17).Little is known about the role of C. jejuni as a triggering agent of GBS in France. In a study of 263 GBS cases in the greater Paris area, France, between 1996 and 2001, serological evidence of recent C. jejuni infection was shown in 22% of patients (16). However, no study has provided direct bacteriological evidence of the role of C. jejuni in French GBS cases, and there are no data on the C. jejuni strains involved. We thus undertook a prospective study on a French cohort of patients with GBS based on the detection of C. jejuni in fecal samples by culture and real-time PCR (RT-PCR).The medical intensive care unit at the Raymond Poincaré Hospital (Garches, France) is a regional reference center for the management of adult patients with GBS. Since November 1999, all admitted GBS cases have been tested for the presence of C. jejuni in fecal samples (rectal swab and/or stool samples) by culture and RT-PCR, and anti-C. jejuni antibodies have been detected by the complement fixation test (CFT). All the patients included in this study were patients with GBS (2) who were admitted between November 1999 and December 2005 and who had (i) at least one fecal sample (rectal swab or stool sample) examined for the presence of C. jejuni by culture and RT-PCR and (ii) measurement of anti-C. jejuni antibodies. The clinical data were collected on inclusion in the study as described previously (16). Serum samples and rectal swabs were taken from patients on admission. The patients'' first stools during their hospitalization were also sampled and studied for C. jejuni culture and RT-PCR. Our ethics committee waived the requirement for informed consent because both the screening of fecal samples for C. jejuni and the measurement of anti-C. jejuni antibodies are routinely carried out and do not affect decisions concerning treatment.Stool samples were cultured with blood-containing Campylosel (bioMérieux, Marcy l''Etoile, France) (November 1999 to May 2002) or Butzler medium (Bio-Rad Laboratories, Hercules, CA) (after May 2002) with or without prior enrichment in Preston selective broth (Oxoid, Basingstoke, Hampshire, England) at 42°C. Rectal swabs were immersed in Preston enrichment broth, which was incubated at 42°C for 24 h before culture on selective medium. Campylobacter isolates were identified to the species level at the French National Reference Center for Campylobacter and Helicobacter. Strains were serotyped with the Penner O scheme (13). Genotyping involved sequence analysis of the short variable region of the flaA gene (flaA SVR) (4) and the Campylobacter Fla database (http://pubmlst.org/campylobacter/flaA/; hosted at the University of Oxford). LOS biosynthesis loci were classified as previously described (10). Campylobacter DNA was amplified from stool and rectal samples by a custom-designed RT-PCR technique using the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany). Bacterial DNA from stool samples was purified by using the QIAamp stool DNA minikit (Qiagen GmbH, Germany), and DNA from rectal samples was purified by using the QIAamp DNA minikit (Qiagen). Purified DNA (2 μl) was subjected to amplification with the 16S DNA primers C16S-F (5′-CTAGCTTGCTAGAACTTAGA-3′) and C16S-R (5′-GTCCACACCTTCCTCCTC-3′). We used the LightCycler FastStart DNA master hybridization probe kit (Roche Diagnostics) with fluorescent hybridization probes LC-Camp16S-A (5′-ACGTATTTAGTTGCTAACGGTTC-fluorescein-3′) and LC-Camp16S-B (5′-LightCycler Red 640-GAGCACTCTAAATAGACTGCCTTC-P-3′), synthesized by Proligo (Paris, France). The specificity of amplification was checked by melting curve analysis (melting peaks for Campylobacter species as follows: C. jejuni and C. coli, 64°C with a shoulder effect at 57°C; C. fetus, 57°C; and C. lari, 54°C). Results are expressed qualitatively, as positive or negative (estimated detection threshold of 2 × 10² CFU/g of feces). Serum antibodies against C. jejuni were assayed with complement fixation tests (CFTs) (Virion\Serion, Würzburg, Germany), using a cutoff titer of 20 (>95% specificity; Virion\Serion). Antibodies (IgM/IgG) against gangliosides GM1, GM2, GD1a, GD1b, and GQ1b were detected by enzyme immunoassays (GanglioCombi; Bühlmann Laboratories AG, Schönenbuch, Switzerland). Statistical analyses were performed with R. 2.8.1 software (The R Foundation for Statistical Computing, Vienna, Austria). Categorical variables were compared using Fisher''s exact tests, and quantitative variables were compared using Wilcoxon tests. All tests were two tailed, and P values of <0.05 were considered significant.We included 237 patients with GBS in this study (Table ​(Table1).1). All patients were assessed for the presence of C. jejuni in fecal samples by culture and RT-PCR (rectal swabs alone, 133 patients; stool samples alone, 23 patients; both samples, 81 patients) and for the presence of serum anti-C. jejuni antibodies with CFT. Sixteen patients (6.8%) gave positive test results with RT-PCR and/or culture; of these, eight were positive by both methods, six were positive by RT-PCR alone, and two were positive by culture alone. C. jejuni was isolated from nine patients, and Campylobacter coli was isolated from one patient. The CFT was positive in 63 patients (26.6%), which included 15 of the 16 patients with positive RT-PCR and/or culture results. Thus, in total, 64 were shown to be associated with C. jejuni (or C. coli) from the 237 patients studied (27%). These cases were more likely to be male, have prodromal diarrhea, be admitted a few days after GBS onset, and have antiganglioside antibodies and a pure motor form than cases with no evidence of recent C. jejuni infection (n = 173) (Table ​(Table11).

TABLE 1.

Characteristics of the 237 patients studied^a

Regulation of Phospholipase C-γ2 and Phosphoinositide 3-Kinase Pathways by Adaptor Proteins In B Lymphocytes

The importance of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)-γ2 in B cell function and development has been highlighted by gene targeting experiments in mice. In fact, these knockout mice exhibit a profound inhibition of proliferative responses upon B cell receptor (BCR) engagement. The molecular connections between these effectors and upstream tyrosine kinases such as Syk have been studied intensively in the past few years. This mechanism involves the action of cytoplasmic adaptor molecules, which participate in forming multicomponent signaling complexes, thereby directing the appropriate subcellular localization of effector enzymes. In addition to these cytoplasmic adaptor proteins, cell surface coreceptors can be viewed as transmembrane adaptor proteins, because coreceptors can also change the localization of effector enzymes, which in turn modulates the BCR-initiated signals.     

Reduced Phosphoinositide 3-Kinase (p110α) Activation Increases the Susceptibility to Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110α) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110α) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult (∼4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice (∼15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110α) and AF.Atrial fibrillation (AF) is a cardiac disorder characterized by uncoordinated atrial activation. It is the most common sustained arrhythmia presenting in cardiology departments worldwide and is associated with substantially increased mortality and morbidity from heart failure, stroke, and thromboembolism.¹ With a growing aging population the incidence of AF is increasing, adding considerably to health care costs.^1,2,3,4 The multifactorial nature of AF and a limited understanding of the molecular mechanisms responsible for the development of AF have greatly limited treatment strategies.Genetically modified mouse models offer a powerful approach to define molecular mechanisms. The very small size of mouse atria, together with the high heart rate (HR), make finding murine AF models difficult, as the potential for re-entry circuits is restricted. Despite these limitations, there are some genetically modified mouse models that are susceptible to AF. These include gene disruption/mutation of connexin40, KCNE1, or nuclear core component NUP155,^5,6,7 overexpression of RhoA,⁸ basic leucine zipper inhibitor protein: JDP2,⁹ angiotensin converting enzyme,¹⁰ tumor necrosis factor α,¹¹ Rac1,¹² and MURC.¹³AF often occurs in combination with heart failure, although the factors that precipitate the onset of AF in patients with (or without) pre-existing heart disease remain unclear.^14,15 We previously demonstrated that phosphoinositide 3-kinase (PI3K, p110α) is a critical regulator of adaptive physiological heart growth,^16,17,18 and that inhibiting PI3K(p110α) accelerates heart failure in a setting of dilated cardiomyopathy (DCM).¹⁹ However, due to the severity of the disease progression in this mouse model (average life span of approximately 40 days¹⁹) it was not possible to perform functional or electrocardiogram (ECG) analyses. The rationale for hypothesizing a link between AF and reduced PI3K(p110α) activity came from multiple lines of evidence. First, we previously demonstrated that decreasing PI3K activation alters gene expression of ion channels in ventricular tissue. Second, several classes of drugs have been reported to induce AF in patients.²¹ The mechanisms considered responsible include adrenergic stimulation and cardiotoxicity.²¹ PI3K(p110α) is a cardioprotective protein that has been shown to inhibit pathological signaling cascades downstream of G protein coupled receptors,¹⁹ thus loss of PI3K(p110α) would be expected to increase the likelihood of cardiotoxicity and activation of signaling proteins downstream of G protein coupled receptors. Third, we previously reported that heat shock protein 70 (Hsp70) expression is elevated in hearts of mice with increased PI3K(p110α) activity and decreased in hearts of mice with decreased PI3K(p110α) activity.²⁰ A number of reports have linked Hsp70 to AF. Patients with high Hsp70 expression levels have a lower incidence of postoperative AF, and an M439T substitution in Hsp70 was associated with an increased risk of postoperative AF.^22,23,24,25 Finally, advanced age and possibly obesity and diabetes are risk factors for the development of AF.^26,27,28 These factors are typically associated with reduced physical activity and insulin resistance. Since PI3K(p110α) is activated in the heart in response to exercise,²⁹ and is a critical molecular signal for insulin, one would predict that PI3K(p110α) activity is generally lower in hearts of obese patients, diabetics, and the elderly.It was also of interest to examine electrical activity in the heart under conditions of reduced PI3K(p110α) activity because of the recent enthusiasm surrounding the development of PI3K(p110α) inhibitors as anticancer agents.^30,31 Uncontrolled activation of the PI3K(p110α) pathway is a critical molecular mechanism by which cancer cells bypass normal growth-limiting controls. However, a challenge in targeting PI3K(p110α) relates to its diverse actions in numerous cell types.³² As noted earlier, PI3K(p110α) is a critical regulator of adaptive physiological heart growth^16,17,18 and we recently demonstrated that inhibiting PI3K(p110α) accelerates heart failure in the compromised heart.¹⁹To reduce PI3K(p110α) activity in a setting of cardiac stress, we genetically crossed a cardiac-specific transgenic (Tg) mouse model of DCM,³³ with a cardiac-specific transgenic mouse expressing a dominant negative mutant of the p110α isoform of PI3K (dnPI3K).¹⁶ Expression of the dnPI3K transgene reduces PI3K activity in cardiac myocytes by approximately 77%.¹⁶ We report here that reduced PI3K(p110α) activity increases the susceptibility to AF in a mouse model of DCM and that PI3K activity is also reduced in atrial appendages from patients with AF compared with those in sinus rhythm.     

Clinical and Molecular Characteristics of 35 Chinese Children with Wiskott–Aldrich Syndrome

Background  Wiskott–Aldrich syndrome (WAS) is a rare primary immunodeficiency disease, with an incidence of 4/1,000,000 live male births. In China, an estimated number of 35 babies with WAS are born each year, but likely many remain undiagnosed. Objectives  The objectives of study were to review the clinical and molecular characteristics of a cohort of Chinese children with WAS and to describe the long-term outcome of those who underwent hematopoietic stem cell transplant (HSCT). Materials and Method  Records of 35 patients diagnosed with WAS during 1991–2008 were reviewed. Genetic diagnosis was established by direct gene sequencing. Results  All patients had classical WAS phenotype. WASP mutations were identified in 33 patients from 29 families. Nine patients underwent HSCT at a mean age of 22.1 months (match-unrelated donor, n = 5; mismatched related donor, n = 2; matched-sibling donor, n = 2). Post-transplant immune hemolytic anemia and thrombocytopenia occurred in three patients with complete resolution. All patients survived without significant long-term complications and had full platelet, T and B lymphocyte recovery within 2 years post-transplant. Conclusion  In the past decade, there has been significant improvement in clinical and genetic diagnosis of WAS in Chinese. We demonstrated excellent long-term survival in patients who underwent HSCT. Early workup for transplant should be advocated for children with classical WAS before they suffer from major disease complications and morbidities. PPW Lee and TX Chen were co-first authors and had equal contributions to the study.     

Adrenocortical Oncocytoma Presenting as Cushing’s Syndrome: An Additional Report of a Paediatric Case

Oncocytomas are tumours predominantly or exclusively composed of oncocytes, cells with granular and eosinophilic cytoplasm filled with mitochondria. Although they can occur in every organ, they are rare in adrenal glands, and in paediatric patients they are even rarer, with only three case reports previously published. We present a preschool child developing Cushing’s syndrome due to an adrenocortical oncocytoma, which was confirmed immunohistochemically with antibodies to the mitochondrial electron complex 2. A 5.8-year-old girl presented with clinical features of Cushing’s syndrome. ACTH-independent hypercortisolism was confirmed biochemically and a left adrenal mass was detected by imaging and removed by laparotomy. Histopathological analysis revealed a tumour composed of more than 95 % of oncocytes, confirmed immunohistochemically with antibodies to subunits A and B of the mitochondrial enzyme succinate dehydrogenase. Using the Lin–Weiss–Bisceglia score system and the reticulin algorithm, this tumour was categorized as a benign adrenocortical oncocytoma. The patient currently has 64 months of follow-up, without any evidence of relapse of symptoms. To our knowledge, we herein present the youngest patient developing an adrenocortical oncocytoma and the first manifestation of Cushing’s syndrome due to this rare neoplasm in paediatric patients. We also emphasize the clinical usefulness of immunohistochemistry to the mitochondrial enzyme succinate dehydrogenase to confirm the oxyphilic nature of adrenocortical oncocytomas.     

PI3 kinase δ is a key regulator of synoviocyte function in rheumatoid arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.     

Phosphoinositide 3-kinase δ is a regulatory T-cell target in cancer immunotherapy

Tumour infiltration by regulatory T (Treg) cells contributes to suppression of the anti-tumour immune response, which limits the efficacy of immune-mediated cancer therapies. The phosphoinositide 3-kinase (PI3K) pathway has key roles in mediating the function of many immune cell subsets, including Treg cells. Treg function is context-dependent and depends on input from different cell surface receptors, many of which can activate the PI3K pathway. In this review, we explore how PI3Kδ contributes to signalling through several major immune cell receptors, including the T-cell receptor and co-stimulatory receptors such as CD28 and ICOS, but is antagonized by the immune checkpoint receptors CTLA-4 and PD-1. Understanding how PI3Kδ inhibition affects Treg signalling events will help to inform how best to use PI3Kδ inhibitors in clinical cancer treatment.     

Complete nucleotide sequence of a Chinese isolate of Grapevine leafroll-associated virus 3 reveals a 5′ UTR of 802 nucleotides

Grapevine leafroll-associated virus 3 (GLRaV-3) is widely spread in China. Here we report, for the first time, the complete nucleotide sequence of the Chinese isolate (LN) of GLRaV-3. The 18,563-nt genomic RNA is the largest of the GLRaV-3 genomes reported to date, with a 5′ untranslated region of 802 nt. Its sequence shares 87.99–98.15 % identity with those of previously reported isolates, and phylogenetic analysis suggested placing isolate LN in group 3, together with another fully sequenced isolate, PL-20.     

Attenuation of phosphoinositide 3-kinase δ signaling restrains autoimmune disease

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn^?/?p110δ^+/KD mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE.     

Central Nervous System Toxoplasmosis with an Increased Proportion of Circulating γδ T Cells in a Patient with Hyper-IgM Syndrome

Hyper-IgM syndrome represents a diverse group of immunodeficiencies characterized by normal or high serum IgM concentrations with decreased or absent IgG, IgA, and IgE. The X-linked form of hyper-IgM syndrome is caused by mutations in the CD40 ligand gene, preventing its expression on activated T cells. The CD40 ligand–CD40 interaction is critical for effective isotype switching and for initiating antigen-specific T cell responses. In addition to recurrent pyogenic infections, patients with the CD40L defect also have opportunistic infections. An increased proportion of circulating T cells, shown to be important early during primary infections, has been demonstrated in numerous infectious diseases including toxoplasmosis. Here, we report a patient with hyper-IgM syndrome and CNS toxoplasmosis, who showed a marked increase in T cells in his peripheral blood and who has responded well to treatment of his toxoplasmosis and to high-dose immunoglobulin replacement therapy.     

Current Trends — Prevention of Acquired Immune Deficiency Syndrome (AIDS) — Report of Inter-Agency Recommendations

Abstract

Clinical and histopathologic findings associated with four diseases in three species of Pacific salmon are described: (1) saprolegniasis in sockeye salmon (Oncorhynchus nerka Walbaum); (2) infection of Chinook salmon (Oncorhynchus tshawytscha Walbaum) by the fungus Phoma herbarum Westend; (3) an unusual case of coldwater disease in coho salmon (Oncorhynchus kisutch Walbaum); and (4) Loma sp. Morrison (Microsporea) infection in chinook salmon. Histochemical techniques with modifications and the importance of histochemistry in supporting the diagnoses are discussed.     

Clinical Aspects and Genetic Analysis of Taiwanese Patients with Wiskott–Aldrich Syndrome Protein Mutation: The First Identification of X-Linked Thrombocytopenia in the Chinese with Novel Mutations

Background  

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and recurrent infections. However, the more than 500 patient mutations described are mainly based on Caucasian and Japanese populations. This study investigated Taiwanese patients with WASP mutations since 1985 as part of a long-term comprehensive study in primary immunodeficiency diseases (PIDs) covering 23 million inhabitants.