Rima Wakim

DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation

The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27+ memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src–kinase Syk–transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

Underlying mechanisms of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: role of OXA-48 and NDM-1 carbapenemases

A recent increase in carbapenem resistance among extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli isolates at a major tertiary care centre in Lebanon prompted the initiation of this study. Consecutive ESBL-producing isolates were tested for resistance to carbapenems, with initial screening by disk diffusion and Etest using ertapenem. The modified Hodge test was also performed. PCR of β-lactamase-encoding genes, including bla(NDM-1), bla(KPC), bla(OXA-48), bla(CTX-M), bla(TEM), bla(SHV), bla(CMY-2) and bla(OXA-1), as well as outer membrane porin genes (ompC and ompF) was performed. Sequencing, efflux pump inhibitor tests and random amplified polymorphic DNA (RAPD) analysis were performed. In total, 14 (2.45%) of 572 K. pneumoniae and 24 (1.07%) of 2243 E. coli were ertapenem-non-susceptible [minimum inhibitory concentration (MIC) ≥0.25 μg/mL]. Resistance to other carbapenems was variable. PCR and sequencing analysis revealed that isolates harboured different β-lactamase genes, including bla(OXA-1), bla(CTX-M-15), bla(TEM-1), bla(CMY-2), bla(OXA-48) and bla(NDM-1). In addition, K. pneumoniae lacked the outer membrane porin-encoding genes, whilst E. coli harboured them with detected mutations. CTX-M-15 was carried on a 90 kb plasmid, whilst OXA-48 was carried on a 70 kb plasmid. Efflux pump inhibition significantly decreased MICs in E. coli. RAPD analysis demonstrated genomic variability. In conclusion, carbapenem resistance in ESBL-producing K. pneumoniae and E. coli is due to the combined effect of β-lactamases with porin impermeability and/or efflux pump activity observed in these organisms, and in a number of isolates is due to the production of the carbapenemase-encoding genes bla(OXA-48) and the newly emerging bla(NDM-1).

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Mutational spectrum of cystic fibrosis in the Lebanese population

BACKGROUND Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians; it is however, considered to be rare in the Arab populations. Reports of the cystic fibrosis transmembrane regulator (CFTR) mutations from Arabs, especially from the Lebanese population, are limited. METHODS Twenty-two unrelated Lebanese families, with at least one child with CF, were studied. DNA extracts from blood samples of patients and parents were screened for CFTR gene mutations. RESULTS Eleven different mutations were identified. Of the 44 alleles studied, the most common mutations were: F508del (34%), N1303K (27%), W1282X (7%), and S4X (7%). Five mutations - not previously reported in the Lebanese population - were identified; these are: S549N, G542X, 2043delG, 4016insG, and R117H-7T. CONCLUSIONS The most common CFTR mutations in addition to five mutations not previously described in the Lebanese population were identified. Identification of CFTR mutations in the Lebanese population is important for molecular investigations and genetic counseling.

Genotypes and serotype distribution of macrolide resistant invasive and non- invasive Streptococcus pneumoniae isolates from Lebanon

BackgroundThis study determined macrolide resistance genotypes in clinical isolates of Streptococcus pneumoniae from multiple medical centers in Lebanon and assessed the serotype distribution in relation to these mechanism(s) of resistance and the source of isolate recovery.MethodsForty four macrolide resistant and 21 macrolide susceptible S. pneumoniae clinical isolates were tested for antimicrobial susceptibility according to CLSI guidelines (2008) and underwent molecular characterization. Serotyping of these isolates was performed by Multiplex PCR-based serotype deduction using CDC protocols. PCR amplification of macrolide resistant erm (encoding methylase) and mef (encoding macrolide efflux pump protein) genes was carried out.ResultsAmong 44 isolates resistant to erythromycin, 35 were resistant to penicillin and 18 to ceftriaxone. Examination of 44 macrolide resistant isolates by PCR showed that 16 isolates harbored the erm(B) gene, 8 isolates harbored the mef gene, and 14 isolates harbored both the erm(B) and mef genes. There was no amplification by PCR of the erm(B) or mef genes in 6 isolates. Seven different capsular serotypes 2, 9V/9A,12F, 14,19A, 19F, and 23, were detected by multiplex PCR serotype deduction in 35 of 44 macrolide resistant isolates, with 19F being the most prevalent serotype. With the exception of serotype 2, all serotypes were invasive. Isolates belonging to the invasive serotypes 14 and 19F harbored both erm(B) and mef genes. Nine of the 44 macrolide resistant isolates were non-serotypable by our protocols.ConclusionMacrolide resistance in S. pneumoniae in Lebanon is mainly through target site modification but is also mediated through efflux pumps, with serotype 19F having dual resistance and being the most prevalent and invasive.

A novel mutation in FOXN1 resulting in SCID: a case report and literature review.

• FOXN1 mutations classically cause T cell lymphopenia, but may also permit non-maternal T cells.

Epidemiology, clinical manifestations, and molecular typing of salmonella typhi isolated from patients with typhoid fever in Lebanon.

The objective of this study was to examine the epidemiology and the clinical manifestations of typhoid fever as well as the susceptibility and strain relatedness of Salmonella typhi isolates in Lebanon from 2006 to 2007. A total of 120 patients with typhoid fever were initially identified from various areas of the country based on positive culture results for S. typhi from blood, urine, stools, bone marrow and/or positive serology. Clinical, microbiological and molecular analysis was performed on cases with complete data available. These results indicated that drinking water was an unlikely mode of transmission of the infection. Despite increasing reports of antimicrobial resistance among S. typhi isolates, the vast majority of these isolates were susceptible to various antibiotic agents, including ampicillin, cephalosporins, quinolones, and trimethoprim/sulfamethoxazole. Molecular analysis of the isolates revealed a predominance of one single genotype with no variation in distribution across the geographical regions.

Antiviral drug susceptibilities of seasonal human influenza viruses in Lebanon, 2008-09 season.

The emergence of antiviral drug‐resistant strains of the influenza virus in addition to the rapid spread of the recent pandemic A(H1N1) 2009 virus highlight the importance of surveillance of influenza in identifying new variants as they appear. In this study, genetic characteristics and antiviral susceptibility patterns of influenza samples collected in Lebanon during the 2008–09 season were investigated. Forty influenza virus samples were isolated from 89 nasopharyngeal swabs obtained from patients with influenza‐like illness. Of these samples, 33 (82.5%) were A(H3N2), 3 (7.5%) were A(H1N1), and 4 (10%) were B. All the H3N2 viruses were resistant to amantadine but were sensitive to oseltamivir and zanamivir; while all the H1N1 viruses were resistant to oseltamivir (possessed H275Y mutation, N1 numbering, in their NA) but were sensitive to amantadine and zanamivir. In the case of influenza B, both Victoria and Yamagata lineages were identified (three and one isolates each, respectively) and they showed decreased susceptibility to oseltamivir and zanamivir when compared to influenza A viruses. Influenza circulation patterns in Lebanon were very similar to those in Europe during the same season. Continued surveillance is important to fully elucidate influenza patterns in Lebanon and the Middle East in general, especially in light of the current influenza pandemic. J. Med. Virol. 82: 1224–1228, 2010.

Dubowitz syndrome: common findings and peculiar urine odor

Background Dubowitz syndrome is a rare, autosomal recessive disorder characterized by intrauterine and postnatal growth retardation, severe microcephaly, psychomotor retardation, hyperactivity, eczema, and characteristic dysmorphic facial features. Although many cases have been reported, the cause of this disease is still unknown. Case We present here the case of a Lebanese girl with Dubowitz syndrome in whom an unpleasant urine odor was persistently reported since birth. Conclusion Although Dubowitz syndrome has been largely described in the medical literature, this is the first time that a peculiar urine odor was reported. This case report adds a new and unusual feature to the numerous findings related to this rare polymorphous syndrome.

Assessment of combination therapy in BALB/c mice injected with carbapenem-resistant Enterobacteriaceae strains.

Monotherapeutic options for carbapenem resistant infections are limited. Studies suggest that combination therapy may be associated with better outcomes than monotherapies. However, this is still controversial. This study assessed, the efficacy of combination therapy against carbapenem resistant Enterobacteriaceae harboring singly various extended spectrum beta lactamase or carbapenemase encoding genes. Thus, four isolates harboring either blaCTXM-15, blaCTXM-15 and blaOXA-48, blaNDM-1, or blaKPC-2 genes were selected for testing. Minimal inhibitory concentration was determined by broth dilution method. Gene transcript levels on single and combined treatments were done in vitro and in vivo by qRT-PCR. Assessment of treatments was done in BALB/c mice according to a specific protocol. As such, the qRT-PCR revealed a significant decrease of transcript levels in all isolates upon using rifampicin or tigecycline, singly or in combination with colistin. However, variable levels were obtained using colistin singly or in combination with meropenem or fosfomycin. In vivo assessment showed that all combinations used were effective against isolates harboring blaCTXM-15, blaOXA-48, and blaNDM-1. Conversely, the most significant combination against the isolate harboring blaKPC-2 gene was colistin with either carbapenem, fosfomycin, or kanamycin. As a conclusion, combination therapy selected based on the type of carbapenemase produced, appeared to be non-toxic and might be effective in BALB/c mice. Therefore, the use of a rationally optimized combination therapy might lead to better results than monotherapy, however, clinical trials are needed for human consumption.