WITHDRAWN: Overview of dengue virus infection in Saudi Arabia

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Isolation of Thermoalkalophilic-?-amylase Producing Bacteria and Optimization of Potato Waste Water Medium for Enhancement of ?-amylase Production

Sixty one thermoalkalophilic bacteria were isolated from soil samples in Saudi Arabia’s southern region. Isolate TA-38, obtained from the Tanomah region, showed the best performance for enzyme production and was submitted for further study. It was identified as Bacillus axarquiensis based on 16S rRNA gene sequencing studies. The feasibility of using potato waste water as a simple and cheap medium for the production of ?-amylase was evaluated compared with starch broth medium. The production of ?-amylase in the potato waste water medium was only 13.8% less than that of the starch medium. Maximum enzyme production was achieved after 48 hours of cultivation at the beginning of the stationary phase at pH 10.0 and 50 0C. The appropriate addition of starch; nitrogen; phosphate; and calcium to potato waste water significantly enhanced the production of ?-amylase. The enzyme production reached a maximum of 64.5 Uml-1 with the potato wastewater adding with 0.5 % starch; 0.4 % yeast extract; 0.04% CaCl2-2H2O and 0.05 % KH2PO4.  The optimization of the potato waste water medium led to an approximately 4.02 fold increase in the production of ?-amylase compared to starch broth medium. Data indicated that the potato waste water contained substrates which could be used by bacterial isolate for the production of ?-amylase production and the developed procedure was cost effective since it requires only a slightly addition of nutrients to the medium. Keywords: Isolation; ?-amylase; 16S rRNA; Production; Potato waste water; Thermoalkaliphilic bacteria

Protocol for a randomised, double-blind, placebo-controlled study of grass allergen immunotherapy tablet for seasonal allergic rhinitis: time course of nasal, cutaneous and immunological outcomes

BACKGROUND: Seasonal Allergic Rhinitis is characterised by inflammation of the nasal mucosa upon exposure to common aeroallergens, affecting up to 20-25 % of the population. For those patients whose symptoms are not controlled by standard medical treatment, allergen specific immunotherapy is a therapeutic alternative. Although several studies have shown changes in immunologic responses as well as long term tolerance following treatment with a sublingual allergy immunotherapy tablet, a detailed time course of the early mechanistic changes of local and systemic T and B cell responses and the effects on B cell repertoire in the nasal mucosa have not been fully examined. METHODS/DESIGN: This is a randomized, double-blind, single-centre, placebo controlled, two arm time course study based in the United Kingdom comparing sublingual allergy immunotherapy tablet (GRAZAX(®), ALK-Abello Horsholm, Denmark) plus standard treatment with placebo plus standard treatment. Up to 50 moderate to severe grass pollen allergic participants will be enrolled to ensure randomisation of at least 44. Further, we shall enrol 20 non-atopic volunteers. Screening will be completed before eligible atopic participants are randomised to one of the two treatment arms in a 1 to 1 ratio. The primary endpoint will be the total nasal symptom score assessed over 60 min following grass pollen nasal allergen challenge after 12 months of treatment. Clinical assessments and/or mechanistic analyses on blood, nasal fluid, brushing and biopsies will be performed at baseline at 1, 2, 3, 4 (coinciding with the peak pollen season), 6 and 12 months of treatment. After 12 months of treatment, unblinding will take place. Those atopic participants receiving active treatment will continue therapy for another 12 months followed by a post treatment phase of 12 months. Assessments and collection of biologic samples from these participants will take place again at 24 and at 36 months from the start of treatment. The 20 healthy, non-atopic controls will undergo screening and one visit only coinciding with the 12 month visit for the atopic participants. DISCUSSION: The trial will end in April 2017. The trial is registered with ClinicalTrials.gov and the trial identifying number is NCT02005627. TRIAL REGISTRATION: Primary Registry: ClinicalTrials.gov, Trial Identifying number: NCT02005627, Secondary identifying numbers: EudraCT number: 2013-003732-72 REC: 13/EM/0351, Imperial College London (Sponsor): 13IC0847, Protocol Version 6.0, Date: 16.05.2014

Local and systemic effects of cat allergen nasal provocation

Background Cat allergen is widely distributed in homes and schools; allergic sensitization is common. Objective To develop a model of cat allergen nasal challenge to establish dose–response and time–course characteristics and investigate local and systemic biomarkers of allergic inflammation. Methods Nineteen cat‐allergic individuals underwent titrated nasal challenge, range 0.243 to 14.6 μg/mL Fel d1, and matched diluent‐only provocation. Clinical response to 8 h was assessed by symptom scores and peak nasal inspiratory flow (PNIF). Nasal fluid was collected using polyurethane sponges and analysed by ImmunoCAP and multiplex assays. Whole blood flow cytometry for basophil surface CD63, CD107a, and CD203c was carried out at baseline and 6 h post‐challenge. Results A dose–response to allergen was seen in symptom scores and PNIF, maximal at 10 000 BU/mL (4.87 μg/mL Fel d1), P < 0.0001 vs. diluent. Nasal fluid tryptase was elevated at 5 min after challenge (P < 0.05 vs. diluent); eotaxin, IL‐4, ‐5, ‐9, and ‐13 were increased at 8 h (P < 0.05 to P < 0.0001 vs. diluent); TSLP was undetectable; IL‐10, IL‐17A, and IL‐33 were unchanged compared to diluent challenge. Nasal fluid IL‐5 and IL‐13 correlated inversely with PNIF after challenge (IL‐5, r = −0.79, P < 0.0001; IL‐13, r = −0.60, P = 0.006). Surface expression of CD63 and CD107a was greater at 6 h than at baseline, both in the presence (both P < 0.05) and absence (CD63, P < 0.01; CD107a, P < 0.05) of in vitro allergen stimulation; no changes were seen on diluent challenge day. Conclusions Cat allergen nasal challenge produces local and systemic Th2‐driven inflammatory responses and has potential as a surrogate outcome measure in clinical trials

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency &gt;3%, and were also present in the mutant library, had fitness levels that were &gt;40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

How safe are the biologicals in treating asthma and rhinitis?

A number of biological agents are available or being investigated for the treatment of asthma and rhinitis. The safety profiles of these biologic agents, which may modify allergic and immunological diseases, are still being elucidated. Subcutaneous allergen immunotherapy, the oldest biologic agent in current use, has the highest of frequency of the most serious and life-threatening reaction, anaphylaxis. It is also one of the only disease modifying interventions for allergic rhinitis and asthma. Efforts to seek safer and more effective allergen immunotherapy treatment have led to investigations of alternate routes of delivery and modified immunotherapy formulations. Sublingual immunotherapy appears to be associated with a lower, but not zero, risk of anaphylaxis. No fatalities have been reported to date with sublingual immunotherapy. Immunotherapy with modified formulations containing Th1 adjuvants, DNA sequences containing a CpG motif (CpG) and 3-deacylated monophospholipid A, appears to provide the benefits of subcutaneous immunotherapy with a single course of 4 to 6 preseasonal injections. There were no serious treatment-related adverse events or anaphylaxis in the clinical trials of these two immunotherapy adjuvants. Omalizumab, a monoclonal antibody against IgE, has been associated with a small risk of anaphylaxis, affecting 0.09% to 0.2% of patients. It may also be associated with a higher risk of geohelminth infection in patients at high risk for parasitic infections but it does not appear to affect the response to treatment or severity of the infection

Allergen Immunotherapy in Children User’s Guide

Allergen immunotherapy is a cornerstone in the treatment of allergic children. The clinical efficiency relies on a well-defined immunologic mechanism promoting regulatory T cells and downplaying the immune response induced by allergens. Clinical indications have been well documented for respiratory allergy in the presence of rhinitis and/or allergic asthma, to pollens and dust mites. Patients who have had an anaphylactic reaction to hymenoptera venom are also good candidates for allergen immunotherapy. Administration of allergen is currently mostly either by subcutaneous injections or by sublingual administration. Both methods have been extensively studied and have pros and cons. Specifically in children, the choice of the method of administration according to the patient's profile is important. Although allergen immunotherapy is widely used, there is a need for improvement. More particularly, biomarkers for prediction of the success of the treatments are needed. The strength and efficiency of the immune response may also be boosted by the use of better adjuvants. Finally, novel formulations might be more efficient and might improve the patient's adherence to the treatment. This user's guide reviews current knowledge and aims to provide clinical guidance to healthcare professionals taking care of children undergoing allergen immunotherapy

Mutational analysis of Rift Valley fever phlebovirus nucleocapsid protein indicates novel conserved, functional amino acids

Rift Valley fever phlebovirus (RVFV; Phenuiviridae, Phlebovirus) is an important mosquito-borne pathogen of both humans and ruminants. The RVFV genome is composed of tripartite, single stranded, negative or ambisense RNAs. The small (S) segment encodes both the nucleocapsid protein (N) and the non-structural protein (NSs). The N protein is responsible for the formation of the viral ribonucleoprotein (RNP) complexes, which are essential in the virus life cycle and for the transcription and replication of the viral genome. There is currently limited knowledge surrounding the roles of the RVFV nucleocapsid protein in viral infection other than its key functions: N protein multimerisation, encapsidation of the RNA genome and interactions with the RNA-dependent RNA polymerase, L. By bioinformatic comparison of the N sequences of fourteen phleboviruses, mutational analysis, minigenome assays and packaging assays, we have further characterised the RVFV N protein. Amino acids P11 and F149 in RVFV N play an essential role in the function of RNPs and are neither associated with N protein multimerisation nor known nucleocapsid protein functions and may have additional roles in the virus life cycle. Amino acid Y30 exhibited increased minigenome activity despite reduced RNA binding capacity. Additionally, we have determined that the N-terminal arm of N protein is not involved in N-L interactions. Elucidating the fundamental processes that involve the nucleocapsid protein will add to our understanding of this important viral protein and may influence future studies in the development of novel antiviral strategies

Functional analysis of the orthobunyavirus nucleocapsid (N) protein

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a tripartite genome consisting of negative sense RNA segments called large (L), medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of 233 amino acids. The N protein encapsidates all three segments to form transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to determine the domain map of BUNV N protein. To investigate residues in BUNV N crucial for its functionality, random and site- specific mutagenesis were performed on a cDNA clone encoding the BUNV N protein. In total, 102 single amino acid substitutions were generated in the BUNV N protein sequence. All mutant N proteins were used in a BUNV minigenome system to compare their activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from parental-like to essentially inactive. The most disruptive mutations were R94A, I118N, W134A, Y141C, L177A, K179I and W193A. Sixty-four clones carrying single substitutions in the BUNV N protein were used in the BUNV rescue system in an attempt to recover viable mutant viruses. Fifty recombinant mutant viruses were rescued and 14 N genes were nonrescuable. The 50 mutant viruses were characterized by: titration, protein labelling, western blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³ -10⁸ pfu/ml, and three different plaque sizes large, medium and small. Protein labelling and western blotting showed that mutations in the N gene did not affect expression of the other viral genes as much as affecting N protein expression. It was demonstrated that single amino acid substitutions could alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F). Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S and S96G were shown to have a ts defect in genome-synthesis (late replication step), while mutant viruses K228T and G230R had a ts defect in antigenome- synthesis (early replication step). Host-restriction experiments were performed using 5 different cell lines (Vero-E6, BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the ratios of intracellular and extracellular particles revealed that only 15% of the total virus particles of mutant Y23A was released as extracellular particles compared to 30% of the parental virus. Fourteen N genes were nonrescuable. They were characterized by (i) their activity in the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact on RNA synthesis. In summary, BUNV N protein was shown to be multi-functional and involved in the regulation of virus transcription and replication, RNA synthesis and assembly, via interactions with the viral L polymerase, RNA backbone, itself or the viral glycoproteins