We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10 12 -10 13 vector genomes͞ ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL͞6 mice and Rag 1 mice. In the immunocompetent C57BL͞6 mice, immunof luorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200-350 ng͞ml) of F.IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunof luorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

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Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly I : C-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-ainduced responses such as interferon-stimulated response element-and AP-1-driven promoter activation, protein kinase R, 29-59-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-a-induced responses. IFN-a treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-a and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-a-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-a-induced expression of 29-59-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.

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Enterovirus 71 (EV71) infections can lead to devastating clinical outcomes in children, with an increasing number of severe cases worldwide. The genetic and antigenic variability of EV71 strains isolated in Taiwan in 1998-2005 was evaluated using partial nucleotide sequence analysis of the VP1 gene and the neutralisation assay. Phylogenetic analyses revealed that most EV71 isolates from the 1998 epidemic belonged to sub-genogroup C2, with a minority belonging to sub-genogroup B4. Between 1999 and 2003, isolates belonging to sub-genogroup B4 predominated, followed by a change to sub-genogroup C4 in 2004 and 2005. Antibodies raised in rabbits or collected from infected patients were able to neutralise EV71 virus stocks at high dilutions, regardless of the sub-genogroup of the virus being challenged. The presence of phylogenetically distinct yet antigenically similar populations of EV71 in Taiwan is of concern in the context of herd immunity and vaccine development.

Food allergy has been traditionally perceived as being rare in Africa. However, the prevalence of other allergic manifestations such as asthma and atopic dermatitis continue to rise in the higher-income African countries. Since the food allergy epidemic in westernized countries has lagged behind that of allergic respiratory conditions, we hypothesize that food allergy is increasing in Africa. This article systematically reviews the evidence for food allergy in Africa, obtained through searching databases including PubMed, Medline, MD Consult, and scholarly Google. Articles are divided into categories based on strength of methodological diagnosis of food allergy. Information was found for 11 African countries: Botswana, Democratic Republic of Congo, Ghana, Kenya, Morocco, Mozambique, Nigeria, South Africa, Tanzania, Tunisia, and Zimbabwe. Most studies reflect sensitization to food or self-reported symptoms. However, a few studies had more stringent diagnostic testing that is convincing for food allergy, mostly conducted in South Africa. Apart from the foods that commonly cause allergy in westernized countries, other regionally significant or novel food allergens may include pineapple (Ghana), okra (Nigeria), and mopane worm (Botswana). Food allergy is definitely an emerging disease in Africa and resources need to be diverted to study, diagnose, treat, and prevent this important disease.