A Fluorescence-Based Assay for N5 -Carboxyaminoimidazole Ribonucleotide Mutase.

The enzyme N5 -carboxylaminoinidazole ribonucleotide (N5 -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5 -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5 -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5 -CAIR synthetase. During our research on identifying inhibitors of N5 -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5 -CAIR mutase.

Isatins Inhibit N5-CAIR Synthetase by a Substrate Depletion Mechanism.

The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N5-CAIR synthetase and N5-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N5-carboxy-5-aminoimidazole ribonucleotide (N5-CAIR). In contrast, vertebrates directly convert AIR to CAIR via the enzyme AIR carboxylase. A high-throughput screen against N5-CAIR synthetase identified a group of compounds with a 2,3-indolinedione (isatin) core that inhibited the enzyme. While initial studies suggested that isatins inhibited the enzyme by a noncompetitive mechanism, here we show that isatins inhibit N5-CAIR synthetase by a substrate depletion mechanism. Unexpectedly, we found that isatin reacts rapidly and reversibly with the substrate AIR. The rate of the reaction is dependent upon the substituents on the phenyl moiety of isatin, with 5- and 7-bromoisatin being faster than 4-bromoisatin. These studies suggest that care should be taken when exploring isatin compounds because the biological activity could be a result of their reactivity.

C1 inhibitor deficiency: 2014 United Kingdom consensus document.

C1 inhibitor deficiency is a rare disorder manifesting with recurrent attacks of disabling and potentially life-threatening angioedema. Here we present an updated 2014 United Kingdom consensus document for the management of C1 inhibitor-deficient patients, representing a joint venture between the United Kingdom Primary Immunodeficiency Network and Hereditary Angioedema UK. To develop the consensus, we assembled a multi-disciplinary steering group of clinicians, nurses and a patient representative. This steering group first met in 2012, developing a total of 48 recommendations across 11 themes. The statements were distributed to relevant clinicians and a representative group of patients to be scored for agreement on a Likert scale. All 48 statements achieved a high degree of consensus, indicating strong alignment of opinion. The recommendations have evolved significantly since the 2005 document, with particularly notable developments including an improved evidence base to guide dosing and indications for acute treatment, greater emphasis on home therapy for acute attacks and a strong focus on service organization.

Quality of life in children with primary antibody deficiency.

Primary antibody deficiency disorders (PADs) can have an excellent outlook if diagnosed early and treated appropriately, but require lifelong treatment with immunoglobulin replacement. Some carry risks of inflammatory complications even with optimal treatment. Quality of life (QoL) and the psychological impact of PADs has been relatively little studied, particularly in children. The purpose of this study was to evaluate QoL and psychological impact in a large group of children affected by a range of PADs, as well as a group with transient hypogammaglobulinemia of infancy (THI). Both parental and, where appropriate, child ratings, were collected using standardised questionnaires (PedsQL and SDQ). Higher rates of psychological difficulties, particularly emotional and peer-relationship difficulties were found in children with PAD when compared with healthy controls. Quality of life was poorer than in healthy controls, and also worse than in children affected by diabetes mellitus. Variations in QoL and the degree of psychological difficulties were found between specific diagnostic groups, with children affected by THI being amongst those with the lowest scores for QoL. Further studies are needed to corroborate and extend these findings, but this study confirms previous findings that primary antibody deficiency has a significant impact on quality of life and psychological well-being, and additionally suggests that the impact varies according to severity of the underlying condition. For those with significant difficulties psychological intervention at an early stage may be beneficial.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Innate immune processes are sufficient for driving cigarette smoke-induced inflammation in mice.

The objective of this study was to characterize the impact of cigarette smoke exposure on lung immune and inflammatory processes. BALB/c and C57BL/6 mice were exposed to cigarette smoke for 4 days (acute) or at least 5 weeks (prolonged). Both mouse strains manifested an inflammatory response after acute smoke exposure, characterized by an influx of neutrophils and mononuclear cells. Multiplex analysis revealed a greater than twofold increase of the cytokines IL-1alpha, -5, -6, and -18, as well as the chemokines monocyte chemotactic protein-1 and -3, macrophage inflammatory protein-1alpha, -beta, and -gamma, -2, -3beta, macrophage defined chemokine, granulocyte chemotactic protein-2, and interferon-gamma-inducible protein-10. In BALB/c mice, neutrophilia persisted after prolonged exposure, whereas C57BL/6 showed evidence of attenuated neutrophilia both in the bronchoalveolar lavage and the lungs. In both mouse strains, cigarette smoke exposure was associated with an expansion of mature (CD11c(hi)/major histocompatibility complex class II(hi)) myeloid dendritic cells; we observed no changes in plasmacytoid dendritic cells. Lymphocytes in the lungs displayed an activated phenotype that persisted for CD4 T cells only after prolonged exposure. In BALB/c mice, T cells acquired T helper (Th) 1 and Th2 effector function after 5 weeks of smoke exposure, whereas, in C57BL/6 mice, neither Th1 nor Th2 cells were detected. In both mouse strains, cigarette smoke exposure led to an accumulation of FoxP3+ T regulatory cells in the lungs. Studies in RAG1 knockout mice suggest that these regulatory cells may participate in controlling smoke-induced inflammation. Acute and prolonged cigarette smoke exposure was associated with inflammation, activation of the adaptive immune system, and expansion of T regulatory cells in the lungs.

Window to His World: Using a Patient's YouTube Channel to Help Diagnose Chronic Mania.

Although chronic mania has been investigated, with several case reports and systematic retrospective cohort studies in the literature, it not a widely recognized entity. No specific definition for chronic mania is provided in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Furthermore, it is challenging to identify patients with chronic mania unless they come to the attention of the legal or medical system. We present the case of a manic patient who was hospitalized and subsequently found to have a YouTube channel that he had been using to promote his self-invented religion for over 2 years. Consent was obtained from the patient to review this YouTube channel for collateral information. From these videos, the patient was seen to be chronically circumstantial in his thought processes, grandiose in his ideas, highly energetic, distractible, preoccupied with religion, and talking with elaborate and rapid speech. A significant improvement in his symptoms was observed after administration of oral risperidone, with his scores on the Young Mania Rating Scale and Brief Psychiatric Rating Scale also showing improvement. To our knowledge, this is the first case in the literature in which an online video-sharing service was used longitudinally to facilitate diagnosis of a mental illness. We suggest that technology has great potential to improve our diagnostic tools, especially for disorders such as chronic mania the diagnosis of which relies primarily on self-report and collateral information.

Tumor necrosis factor-alpha inhibits epithelial differentiation and morphogenesis in the mouse metanephric kidney in vitro.

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has diverse actions both within and outside the immune system and has been implicated in the etiology of a wide range of pathological conditions. Evidence is accumulating that it may also have important roles in the normal development of the embryo. In this study we demonstrated that the addition of recombinant TNF-alpha to metanephric organ culture induced a dose dependent and reversible decrease in growth and development, with inhibition of ureteric bud branching and nephron formation beyond the condensate stage and despite appropriate expression of the transcription factor pax-2. TNF-alpha also increased the point prevalence of apoptosis after only 1 day of culture. We also noted that macrophages were present in renal rudiments at the inception of nephrogenesis and their numbers significantly increased during the culture period. This effect was enhanced by TNF-alpha. We have also demonstrated expression of mRNAs for TNF-alpha and its receptors in whole mouse metanephroi from the inception of renal development. TNF-alpha protein was also detected, predominantly at mesenchymal/epithelial interfaces. In addition, TNF-alpha mRNA and protein were expressed by clonal renal mesenchymal cells in vitro, suggesting that these cells are a source of TNF-alpha in vivo.

Impact of viral reactivations in the era of pre-emptive antiviral drug therapy following allogeneic haematopoietic SCT in paediatric recipients.

While pre-emptive rituximab therapy for EBV has substantially reduced the incidence of post-transplant lymphoproliferative disorder, following allogeneic haematopoietic SCT (HSCT), cytomegalovirus (CMV) and adenovirus (ADV) still contribute to significant morbidity and mortality after HSCT. We therefore aimed to identify high-risk children who could benefit from recent advances in virus-specific immunotherapy, define the impact of viral reactivations on survival and estimate the economic burden of pre-emptive antiviral drug therapy. Between 2005 and 2010, prospective monitoring of 291 paediatric HSCT procedures revealed that reactivation of CMV (16%), ADV (15%) and EBV (11%) was frequent during period of CD4 T-cell lymphopenia (0.15 × 10(9) L(-1); P<0.05). We report significant risk factors for reactivation, most notably the use of serotherapy and development of GVHD (grade II) in the presence of pre-existing infection (ADV) or donor and/or recipient seropositivity (CMV, EBV). Most interestingly, CMV and ADV viraemia were the major independent predictors of mortality (P<0.05). CMV, ADV or EBV viral reactivation caused prolonged hospitalization (P<0.05), accounted for 15% of all mortality and substantially increased the cost of transplantation by ∼£22 500 ($34 000). This provides an economic rationale for targeting high-risk HSCT recipients with interventions such as virus-specific cell therapy.