Rare mendelian primary immunodeficiency diseases associated with impaired NF-κB signaling.

Mendelian primary immunodeficiency diseases (MPIDs) are rare disorders affecting distinct constituents of the innate and adaptive immune system. Although they are genetically heterogeneous, a substantial group of MPIDs is due to mutations in genes affecting the nuclear factor-κB (NF-κB) transcription pathway, essential for cell proliferation and cell survival and involved in innate immunity and inflammation. Many of these genes encode for crucial regulatory components of the NF-κB pathway and their mutations are associated with immunological and developmental signs somehow overlapping in patients with MPIDs. At present, nine different MPIDs listed in the online mendelian inheritance in man (OMIM) are caused by mutations in at least nine different genes strictly involved in the NF-κB pathway that result in defects in immune responses. Here we report on the distinct function of each causative gene, on the impaired NF-κB step and more in general on the molecular mechanisms underlining the pathogenesis of the disease. Overall, the MPIDs affecting the NF-κB signalosome require a careful integrated diagnosis and appropriate genetic tests to be molecularly identified. Their discovery at an ever-increasing rate will help establish a common therapeutic strategy for a subclass of immunodeficient patients.

Nuclear factor-kappa-B-inhibitor alpha (NFKBIA) is a developmental marker of NF-κB/p65 activation during in vitro oocyte maturation and early embryogenesis.

BACKGROUND: The oocyte-to-embryo transition (OET) requires a co-ordinated transcriptional programme acting through evolutionarily conserved events, and transcription factors (TFs) are known to control these processes. Here, we focus on nuclear factor (NF)-κB, a TF involved in several cellular processes, studying NFκB-inhibitor (NFKBIA) mRNA and its protein product, IκBα, during OET. NFKBIA and IκBα are part of a regulatory loop, as IκBα is the major down-regulator of NF-κB activation while NFKBIA transcription is activated by NF-κB. METHODS AND RESULTS: We found a dynamic correlation between NFKBIA transcript, expression of IκBα-protein and activation of NF-κB/p65 in bovine oocyte and embryo. During the transition from immature to in vitro matured bovine oocyte, we observed a decrease in maternal NFKBIA mRNA and a parallel increase of the IκBα-protein (both P < 0.05). In the embryo, NFKBIA neo-synthesis is activated as a consequence of embryo genome activation (EGA), and IκBα decreases. NF-κB/p65-binding activity was detectable at low levels in immature oocyte, disappeared in dormant metaphase II oocyte and was strong in the embryo, during embryonic NFKBIA synthesis. The level of NF-κB/p65 DNA binding correlates with the timing of meiotic silencing during bovine oocyte maturation and embryonic transcription reprogramming. CONCLUSIONS: The IκBα/NF-κB circuit appears to be a tightly stage-controlled mechanism that could govern OET, being activated at EGA. Our findings represent the first characterization of NFKBIA and IκBα as maternal effectors in both the bovine oocyte and embryo. We suggest a role for NFKBIA as a marker of NF-κB/p65 activation in the human oocyte and early embryo.

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high performance [correction of power] liquid chromatography method.

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.