Disease course and treatment effects of a JAK inhibitor in a patient with CANDLE syndrome.

BACKGROUND: CANDLE syndrome (an acronym for Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature) is a recently described rare autosomal recessive disorder charaterized by systemic autoinflammation. Clinical manifestations include presentation in the first year of life, episodes of fever accompanied by erythematous skin lesions, progressive lipodystrophy, violaceous periorbital swelling and failure to thrive. This syndrome is caused by loss of function mutations and malfunction of the immunoproteasome complex. Most patients have biallelic mutations in the PSMB8 gene that encodes the ß5i catalytic subunit of the immunoproteasome. Examples of digenic inheritance have been also described in CANDLE. CANDLE patients have strong type I interferon gene expression signature and they are responsive to treatment with JAK inhibitors. However, possible serious side-effects remain a concern. Here, we report another patient with CANDLE whose disease activity was well controlled by the treatment with baricitinib. CASE PRESENTATION: We report a Bulgarian patient of the Turkish ancestry who carries biallelic mutations in the PSMB8 gene: p.Ala92Val and p.Lys105Gln. The pathogenic variant p.Ala92Val has not been previously described in patients with CANDLE. We also comment on the unusual feature in this patient, nephrolithiasis, that has not been described in other patients, however it might be related to the positive family history for kidney stones. We have treated the patient with the JAK inhibitor baricitinib for the past year and we observed a significant amelioration of his inflammatory episodes, skin and joint manifestations, and improvements in physical activities and growth. The treatment with glucocorticoids (GC) was completely discontinued. No side effects have been observed, however they remain in consideration for a life-long therapy of this disease. CONCLUSIONS: CANDLE should be suspected in patients with early-onset systemic inflammatory disease and prominent skin manifestations. Molecular testing can confirm the clinical diagnosis and is very important in guiding therapies. Treatment with JAK inhibitors is highly efficacious and appears to be safe in children with CANDLE and other intereforonopathies.

Clinical and laboratory correlates of lung disease and cancer in adults with idiopathic hypogammaglobulinaemia.

Idiopathic hypogammaglobulinaemia, including common variable immune deficiency (CVID), has a heterogeneous clinical phenotype. This study used data from the national UK Primary Immune Deficiency (UKPID) registry to examine factors associated with adverse outcomes, particularly lung damage and malignancy. A total of 801 adults labelled with idiopathic hypogammaglobulinaemia and CVID aged 18-96 years from 10 UK cities were recruited using the UKPID registry database. Clinical and laboratory data (leucocyte numbers and serum immunoglobulin concentrations) were collated and analysed using uni- and multivariate statistics. Low serum immunoglobulin (Ig)G pre-immunoglobulin replacement therapy was the key factor associated with lower respiratory tract infections (LRTI) and history of LRTI was the main factor associated with bronchiectasis. History of overt LRTI was also associated with a significantly shorter delay in diagnosis and commencing immunoglobulin replacement therapy [5 (range 1-13 years) versus 9 (range 2-24) years]. Patients with bronchiectasis started immunoglobulin replacement therapy significantly later than those without this complication [7 (range 2-22) years versus 5 (range 1-13) years]. Patients with a history of LRTI had higher serum IgG concentrations on therapy and were twice as likely to be on prophylactic antibiotics. Ensuring prompt commencement of immunoglobulin therapy in patients with idiopathic hypogammaglobulinaemia is likely to help prevent LRTI and subsequent bronchiectasis. Cancer was the only factor associated with mortality. Overt cancer, both haematological and non-haematological, was associated with significantly lower absolute CD8(+) T cell but not natural killer (NK) cell numbers, raising the question as to what extent immune senescence, particularly of CD8(+) T cells, might contribute to the increased risk of cancers as individuals age.

Spectrum and management of complement immunodeficiencies (excluding hereditary angioedema) across Europe.

INTRODUCTION: Complement immunodeficiencies (excluding hereditary angioedema and mannose binding lectin deficiency) are rare. Published literature consists largely of case reports and small series. We collated data from 18 cities across Europe to provide an overview of primarily homozygous, rather than partial genotypes and their impact and management. METHODS: Patients were recruited through the ESID registry. Clinical and laboratory information was collected onto standardized forms and analyzed using SPSS software. RESULTS: Seventy-seven patients aged 1 to 68 years were identified. 44 % presented in their first decade of life. 29 % had C2 deficiency, defects in 11 other complement factors were found. 50 (65 %) had serious invasive infections. 61 % of Neisseria meningitidis infections occurred in patients with terminal pathway defects, while 74 % of Streptococcus pneumoniae infections occurred in patients with classical pathway defects (p < 0.001). Physicians in the UK were more likely to prescribe antibiotic prophylaxis than colleagues on the Continent for patients with classical pathway defects. After diagnosis, 16 % of patients suffered serious bacterial infections. Age of the patient and use of prophylactic antibiotics were not associated with subsequent infection risk. Inflammatory/autoimmune diseases were not seen in patients with terminal pathway, but in one third of patients classical and alternative pathway defects. CONCLUSION: The clinical phenotypes of specific complement immunodeficiencies vary considerably both in terms of the predominant bacterial pathogen, and the risk and type of auto-inflammatory disease. Appreciation of these phenotypic differences should help both immunologists and other specialists in their diagnosis and management of these rare and complex patients.

Deep fungal dermatitis caused by the Chrysosporium anamorph of Nannizziopsis vriesii in captive coastal bearded dragons (Pogona barbata).

Deep fungal dermatitis caused by the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) was diagnosed in a group of coastal bearded dragons (Pogona barbata). The outbreak extended over a 6-month period, with four of six lizards from the same zoological outdoor enclosure succumbing to infection. A fifth case of dermatomycosis was identified in a pet lizard originally sourced from the wild. Diagnosis of infection with the CANV was based on similar clinical signs and histopathology in all animals and confirmed by culture and sequencing of the fungus from one animal. This is the first report of the CANV causing disease in a terrestrial reptile species in Australia and the first in the coastal bearded dragon.

Predicting Soybean Rust Incursions into the North American Continental Interior Using Crop Monitoring, Spore Trapping, and Aerobiological Modeling.

Between 2005 and 2009, millions of U.S. and Canadian soybean acres that would have received fungicide application remained untreated for soybean rust due to information disseminated through the Integrated Pest Management Pest Information Platform for Extension and Education (ipmPIPE), increasing North American producers' profits by hundreds of millions of dollars each year. The results of our analysis of Phakopsora pachyrhizi urediniospores in rain collections, aerobiology model output, and observations of soybean rust spread in 2007 and 2008 show a strong correspondence between spore collections and model predictions for the continental interior of North America, where soybean is an important crop. The analysis suggests that control practices based on up-to-date maps of soybean rust observations and associated commentary from Extension Specialists delivered by the ipmPIPE may have suppressed the number and strength of inoculum source areas in the southern states and retarded the northward progress of seasonal soybean rust incursions into continental North America. The analysis further indicates that spore trapping and aerobiological modeling can reduce our reliance on the costly Sentinel Plot Network while maintaining the effectiveness of the ipmPIPE system for soybean rust management.

Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency.

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαß+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

Cholesterol dependence of varicella-zoster virion entry into target cells.

The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-beta-cyclodextrin (M beta CD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by M beta CD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.

First Report of Puccinia lagenophorae on Common Groundsel (Senecio vulgaris) in Canada.

Rust disease on common groundsel was independently collected from two backyard gardens in Alberta, Canada during 2005, the first on September 11 in Sherwood Park (53.542°N, 113.262°W) and the second on September 18 in Edmonton (53.463°N, 113.593°W). Leaves of each specimen had clusters of orange, cup-shaped aecia, bordered by recurved peridia, the principal macroscopic signs of disease. Infected plants had twisted stems and deformed leaves. Spores of isolates from the two locations were (mean diameter [± s.d.; range]) 14.6 (± 1.4; 11.4 to 18.9) × 12.5 (± 1.1; 9.1 to 16.2) µm, orange, oval or angular, and many had refractive granules (3). Genomic DNA was extracted from small leaf pieces with multiple aecia, and the complete internal transcribed spacer (ITS) region of the rust was sequenced from PCR products. The sequences determined for a representative specimen from each location were identical, including two areas of ambiguity in the ITS1 spacer region. At position 7 were two overlapping peaks (A and C), and near position 130, sequencing failed because of a suspected insertion/deletion in some ITS copies. Difficulties of sequencing through this cytosine-rich area were reported by Littlefield et al. (3). Data from cloned PCR products confirmed the presence of two ITS genotypes in each DNA extract, one identical to a sequence published for Puccinia lagenophorae on Senecio vulgaris from the United Kingdom (GenBank Accession No. AY808060 (2), and the other identical to a sequence from the United States (GenBank Accession No. AY852264) (3). They differ by an A/C transversion at position 7 and an indel, an 8/9 base poly-C run beginning at position 130. Telia and teliospores were not observed in any of the 2005 samples (some collected as late as November) or in the 2006 Edmonton site samples. Identification of the pathogen as P. lagenophorae was based on host plant symptoms (3) and molecular characters. The original source of inoculum for these infections is unknown, but on December 5, 2006, diseased specimens with sporulating aecia were found beneath 45 cm of snow at the Edmonton location, in a garden area that had not been weeded during the summer. There is reported evidence that teliospores are not functional and that P. lagenophorae overwinters on infected plants that develop aecia in the spring (1). Specimens have been deposited at the Arthur Herbarium, Purdue University, West Lafayette, IN (Vouchers PUR N5414-N5417) and the National Mycological Herbarium of Canada, Ottawa, ON (Vouchers DAOM 237844, 237845, 237961, 237962, 237982, and 237990). The two cloned variants of the ITS sequence were deposited in GenBank (Accession Nos. EF212446 and EF212447). To our knowledge, this is the first report of groundsel rust caused by P. lagenophorae in Canada (G. Barron, personal communication, has images from Guelph in 2004 but no specimens were examined or preserved). Groundsel rust has been found at several locations in the United States (3) and has been reported on more than 60 species in several genera (4). Questions remain about the amount of damage that P. lagenophorae will cause to groundsel in North America and whether it will affect native Senecio species and their relatives. References: (1) J. Frantzen and H. Müller-Schärer. Plant Pathol. 48:483, 1999. (2) B. Henricot and G. Denton. Plant Pathol. 54:242, 2005. (3) L. Littlefield et al. Ann. Appl. Biol. 147:35, 2005. (4) M. Scholler. J. Plant Dis. Prot. 105:239, 1998.

The role of the trans-Golgi network in varicella zoster virus biology.

The task of assembling nascent virions presents a formidable challenge to large, enveloped DNA viruses such as varicella zoster virus (VZV). After parasitising the host cell's compartmentalised biosynthetic machinery, viral constituents must be brought together in appropriate proportions for packaging and export. Recent evidence places the trans-Golgi network (TGN) in an orchestrating role with respect to the assembly, envelopment and egress of herpesviruses. This role accords with known functions of the TGN in the uninfected cell. The targeting of viral glycoproteins to the TGN appears to provide a crucial platform for viral assembly. Tegument proteins, interacting with the cytoplasmic domains of glycoproteins, in turn recruit nucleocapsids to the developing supramolecular array. Molecular studies are continually refining understanding of these processes, building upon elegant electron microscopic data. Knowledge of VZV's use of endogenous trafficking pathways from the TGN sheds light on important aspects of viral behaviour in vitro and in vivo.

Morphology and phylogenetic placement of Endoconidioma, a new endoconidial genus from trembling aspen.

Endoconidioma populi gen. et sp. nov. is described from black subicula on twigs of trembling aspen, Populus tremuloides, in Alberta, Canada. Pycnidium-like conidiomata are produced on twigs and in culture, but, unlike pycnidia, conidiomata of E. populi have a closed peridium and a locule filled with conidiogenous cells that form conidia endogenously. These endoconidia are hyaline, unicellular and released by the dissolution of the peridial cell wall. In addition to endoconidia, mostly two-celled conidia that form blastically from undifferentiated hyphae occur often in culture but are observed only occasionally on Populus twigs. No coelomycetous taxa have been reported to produce endoconidia, and both the morphological features and DNA sequence data demonstrate that Endoconidioma is distinct from the previously established endoconidial genera. Parsimony analyses of portions of the nuclear ribosomal RNA gene (SSU and ITS) suggest that Endoconidioma is closely related phylogenetically to members of the Dothideales and allied anamorphs in Hormonema and Kabatina.

Comparative morphology and phylogenetic placement of two microsclerotial black fungi from Sphagnum.

Capnobotryella renispora and Scleroconidioma sphagnicola form black, irregularly shaped microsclerotia that are indistinguishable in gross morphology on leaves of Sphagnum fuscum. In culture, microsclerotia of these fungi were similar, in that mature component cells possessed thick, highly melanized cell walls, poorly defined organelles, large lipid bodies and simple septa. They were different in morphogenesis, in the way their component cells were organized and in disseminative propagules. Microsclerotia of S. sphagnicola formed phialidic conidiogenous cells on their surface, whereas in C. renispora, adjacent cells in mature microsclerotia often separated from each other by septum schizolysis and formed chlamydospores. The identification of C. renispora from Sphagnum is provisional despite a 100% ITS sequence match with data for a culture derived from the type strain. No holoblastic, reniform conidia typical of the species were formed in nature or in culture, and the SSU sequence for a separately preserved culture of the ex-type strain was markedly divergent. Parsimony analyses of nuclear ribosomal DNA sequences showed that these two fungi were related to separate orders of Dothideomycetes. Both SSU and ITS data supported a close relationship for S. sphagnicola to the Dothideales sensu stricto, while the closest ITS match was to Rhizosphaera spp. In the SSU analyses, C. renispora was nested within the Capnodiales.

First Report of Brown Rot in Wine Grapes Caused by Monilinia fructicola in Canada.

A survey was conducted in 2001 and 2002 to determine incidence of fruit pathogens in wine grapes (Vitis vinifera), an important crop in the southern interior of British Columbia (BC), Canada. Grape clusters were sampled every 2 weeks from June to October at eight vineyard sites located from Osoyoos in the south to Kelowna, approximately 100 km to the north. In the laboratory, the berry clusters were surface disinfested for 0.5 min in 70% ethanol, followed by 1 min in 0.5% sodium hypochlorite, and rinsed twice in sterile distilled water. The berries were placed on potato dextrose agar (PDA) amended with 15 ml/liter of 85% lactic acid and incubated at 20°C for 1 week. During the 2002 survey, a fungus resembling Monilinia fructicola (G. Wint.) Honey was observed sporulating on immature 'Pinot noir' grapes from Kelowna that were sampled on 14 August. Later in the growing season, a similar fungus was detected on 'Riesling' grapes from Summerland sampled on 11 September. There was no evidence of brown rot near the vineyard in Kelowna, but diseased stonefruit were present near the vineyard in Summerland. Subsequent identification of the fungus from 'Riesling' as M. fructicola was based on morphological characters and DNA sequence data for the internal transcribed spacer (ITS) regions of the nuclear ribosomal rRNA genes. The sequenced isolate was deposited in the Canadian Collection of Fungus Cultures as DAOM 231119, and the ITS sequence was accessioned in GenBank as AY289185. Colony growth on PDA was rapid and in concentric rings with the colony margin complete, microconidia abundant, and macroconidia 12 to 13 µm long. Macroconidia germinated with a long germ tube before branching. These characteristics distinguished this fungus from M. laxa, a closely related species that is slow growing with lobed colony margins, produces few microconidia, and germ tubes that branch close to the conidium (1). The complete ITS sequence for DAOM 231119 was a 100% match to other sequences deposited for M. fructicola (Z73777, AF010500, and U21815). On the basis of comparisons of available data, ITS sequences for M. fructicola (three complete ITS, seven partial ITS) and M. laxa (8 complete ITS, 10 partial ITS) differed consistently at four nucleotide positions. The fungus identified as M. fructicola was tested for pathogenicity on mature surface-sterilized 'Pinot noir' and 'Riesling' grapes. Under humid conditions, buff-colored sporodochia bearing conidia developed over the surface of the infected berries. This indicates that M. fructicola can cause decay of wine grapes and could be confused with bunch rot caused by Botrytis cinerea. Previously, M. fructicola was reported on grapes in Oklahoma, but likely these grapes were not Vitis vinifera (2). To our knowledge, this is the first report of brown rot caused by M. fructicola on wine grapes in North America. References: (1) L. R. Batra. World Species of Monilinia (Fungi): Their Ecology, Biosystematics and Control. Mycologia Memoir No. 16. Gerbrüder Borntraeger, Berlin/Stuttgart, 1991. (2) D. A. Preston. Host Index of Oklahoma Plant Diseases, Tech. Bull. No. 21. Oklahoma Agricultural and Mechanical College, Agricultural Experiment Station, Stillwater, 1945.

Recombinant modified vaccinia virus Ankara efficiently restimulates human cytotoxic T lymphocytes in vitro.

The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.

Memory CD8+ T cells in HIV infection.

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.

Rapid effector function in CD8+ memory T cells.

The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon gamma (IFN-gamma) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-gamma release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I-restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

MYCORRHIZAE AND MYCORRHIZAL FUNGI OF CALYPSO BULBOSA.

The seasonal development and fungal endophytes of the mycorrhizal roots of Calypso bulbosa (L.) Oakes were studied using fresh collections from Alberta and herbarium specimens from six herbaria. Typically, a single pair of mycorrhizal roots are produced at the base of a single corm each growing season. No evidence was found to support the putative existence of a coralloid rhizome in this species. The pattern of fungal infection, peloton formation, and breakdown is similar to other nonsaprophytic, terrestrial orchids. The endophytic fungi of a single mycorrhizal root can belong to a number of different fungal taxa. Fungi isolated and described include Rhizoctonia anaticula Currah, Thanatephorus pennatus Currah, Leptodontidium orchidicola Sigler and Currah, Phialocephala fortinii Wang and Wilcox, and two unnamed isolates of Rhizoctonia.