High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors.     

Regulation of RANKL and OPG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg-gingipains

Porphyromonas gingivalis is highly implicated in the pathogenesis of periodontitis, which is characterized by the destruction of periodontal connective tissues and the supporting alveolar bone. Receptor Activator of NF-kappaB Ligand (RANKL) stimulates bone resorption, whereas osteoprotegerin (OPG) blocks its action, and this bi-molecular system is implicated in periodontitis. The aim of this work was (a) to investigate the regulation of RANKL and OPG gene expression in human periodontal ligament (PDL) cells and gingival fibroblasts (GF), in response to P. gingivalis culture supernatants, by quantitative real-time PCR and (b) to attempt to identify putative virulence factors involved in this process. The results indicated that P. gingivalis induced RANKL and reduced OPG mRNA expression by the studied cells, resulting in an increased RANKL/OPG expression ratio. Heat-inactivation of P. gingivalis resulted in significant reduction of RANKL mRNA expression. A Lys-gingipain mutant strain did not affect, whereas an Arg-gingipain mutant strain further enhanced RANKL mRNA expression, compared to their parental wild-type strain. In conclusion, P. gingivalis up-regulates the RANKL/OPG expression ratio in GF and PDL cells, denoting an enhanced osteoclastogenic potential by the cells. The component mainly responsible for RANKL induction appears to be proteinaceous, and it may be regulated by the Arg-gingipains.     

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants

Rationale An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized.
Methods Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors.
Results Following gel filtration, 75 ± 7% of the applied protein and 76 ± 16% ( n =3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 ± 2%; n =3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ∼20 kDa and a range of 13–25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 ± 43% when measured with a pool of serum ( n =2) and 66 ± 7% when measured with individual sera ( n =4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 ± 2% ( n =2) of the original extract when assayed with the serum pool and 10 ± 4% ( n =3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins.
Conclusions Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.     

Stimulation of amylase and sialic acid releases from dog submandibular gland slices by pilocarpine or high K+ medium: a possible role of calmodulin for their releases

The mechanism of amylase and sialic acid releases stimulated by pilocarpine or high K+ medium was investigated in the slices of dog submandibular glands. The release of both amylase and sialic acid was dose-dependently increased by pilocarpine and a considerable release was observed at pilocarpine concentrations of more than 1 microM. Similar effects were observed when K+ concentration in the medium was increased and the maximal response was observed at 75 mM K+. The release of amylase and sialic acid by pilocarpine or K+ considerably decreased by removing Ca2+ from the medium and the slices. The release of amylase in the Ca2+-deficient slices was nearly recovered by the addition of 2.5 and 5.0 mM Ca2+, whereas that of sialic acid was recovered by only 60-75%. Ca2+ inhibitors, La3+ and verapamil, and calmodulin inhibitors, trifluoperazine, prenylamine, and W-7, significantly inhibited the release of amylase and sialic acid induced by the stimulants. These results suggest that the release of amylase and sialic acid stimulated by pilocarpine or K+ is dependent on the presence of Ca2+, and that the activation of calmodulin is involved in the process of the release.     

Pseudohyphal differentiation defect due to mutations in GPCR and ammonium signaling is suppressed by low glucose concentration: a possible integrated role for carbon and nitrogen limitation

In response to carbon and/or nitrogen limitation, diploid cells of Saccharomyces cerevisiae either sporulate or develop pseudohyphae. Although the signal transduction pathways leading to these developmental changes have been extensively studied, how nutritional signals are integrated is not clearly understood. Results of this study indicate that reducing glucose concentration from 2% (SLAD) to 0.05% (SLALD) causes an increase in the magnitude of filamentation as well as a discernible reduction in the time required for pseudohyphal development. Further, the pseudohyphal defect of gpa2, gpr1and gpa2gpr1 but not the mep2 mutant strain is overcome on SLALD. Low glucose also induced pseudohyphae in mep2gpr1 but not mep2gpa2 strain suggesting that GPR1 inhibits pseudohyphae by inhibiting GPA2 function. Accordingly, deleting GPA2 in mep2gpr1 mutant abrogated pseudohyphae formation in SLALD. Further, replenishment of glucose suppressed pseudohyphal differentiation in wild-type cells grown in SLAD medium. However, in SLALD, glucose replenishment suppressed the filamentation response of gpa2 mutants but not that of strains carrying the wild-type GPA2. Increased trehalose levels correlated with decreased pseudohyphae formation. Results of this study demonstrate that filamentation in response to nitrogen limitation occurs as glucose becomes limiting.     

Enrichment of nucleofected primary human CD4+ T cells: a novel and efficient method for studying gene function and role in human primary T helper cell differentiation

Identification of key factors mediating the differentiation of naïve CD4⁺ T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4⁺ T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2K^k cell surface marker followed by selection of H-2K^k positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2K^k positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4⁺ T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.     

Comparing COVID-19 vaccines for their characteristics,efficacy and effectiveness against SARS-CoV-2 and variants of concern: a narrative review

BackgroundVaccines are critical cost-effective tools to control the coronavirus disease 2019 (COVID-19) pandemic. However, the emergence of variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may threaten the global impact of mass vaccination campaigns.AimsThe objective of this study was to provide an up-to-date comparative analysis of the characteristics, adverse events, efficacy, effectiveness and impact of the variants of concern for 19 COVID-19 vaccines.SourcesReferences for this review were identified through searches of PubMed, Google Scholar, BioRxiv, MedRxiv, regulatory drug agencies and pharmaceutical companies' websites up to 22nd September 2021.ContentOverall, all COVID-19 vaccines had a high efficacy against the original strain and the variants of concern, and were well tolerated. BNT162b2, mRNA-1273 and Sputnik V after two doses had the highest efficacy (>90%) in preventing symptomatic cases in phase III trials. mRNA vaccines, AZD1222, and CoronaVac were effective in preventing symptomatic COVID-19 and severe infections against Alpha, Beta, Gamma or Delta variants. Regarding observational real-life data, full immunization with mRNA vaccines and AZD1222 seems to effectively prevent SARS-CoV-2 infection against the original strain and Alpha and Beta variants but with reduced effectiveness against the Delta strain. A decline in infection protection was observed at 6 months for BNT162b2 and AZD1222. Serious adverse event rates were rare for mRNA vaccines—anaphylaxis 2.5–4.7 cases per million doses, myocarditis 3.5 cases per million doses—and were similarly rare for all other vaccines. Prices for the different vaccines varied from $2.15 to $29.75 per dose.ImplicationsAll vaccines appear to be safe and effective tools to prevent severe COVID-19, hospitalization, and death against all variants of concern, but the quality of evidence greatly varies depending on the vaccines considered. Questions remain regarding a booster dose and waning immunity, the duration of immunity, and heterologous vaccination. The benefits of COVID-19 vaccination outweigh the risks, despite rare serious adverse effects.     

The profiles of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood mononuclear cells from house-dust-mite-allergic patients: a role for IL-6 in allergic disease

We have developed a model to measure cytokine production by peripheral blood mononuclear cells (PBMC) in vitro. In this report, we examine the production of interleukin-2 (IL-2), IL-6, and interferon-gamma (IFN-γ) by PBMC of house-dust-mite ( Dermatophagoides pteronyssinus )-allergic subjects. When stimulated with specific allergen ( D. pteronyssinus ), PBMC of patients produced significant levels of IL-2 and high levels of IL-6, but little or no IFN-γ. Nonatopic control PBMC also produced IL-6, although at lower levels, but no IL-2 or IFN-γ. A ubiquitous antigen, streptokinase/streptodornase (SKSD), induced high levels of IL-2 in patients, but only low levels of IFN-γ and IL-6. Nonatopic controls produced similar levels of IL-2 and IL-6, but high levels of IFN-γ to SKSD. IL-2 and IFN-γ levels induced by the T-cell mitogen phytohaemagglutinin (PHA) were similar in patient and control groups, but IL-6 levels were significantly lower in the patients. IgE synthesis in vitro was shown only in atopic PBMC cultures stimulated with specific allergen. The major points can be summarized as 1) IL-2 production by atopic patients in response to allergen; 2) IL-6 production to allergen by both atopic and nonatopic patients, but significantly increased in atopic patients; and 3) defective IFN-γ production by atopic patients to both allergen and antigen. These findings suggest that IL-6 may be important in the immune response to inhalent allergens such as D. pteronyssinus , possibly by creating a cytokine environment favourable to a T^H2 response, and that atopic patients exhibit a generalized defect of IFN-γ production, not related to the response to allergen.     

Novel KCNQ2 and KCNQ3 Mutations in a Large Cohort of Families with Benign Neonatal Epilepsy: First Evidence for an Altered Channel Regulation by Syntaxin‐1A

Mutations in the KCNQ2 and KCNQ3 genes encoding for K_v7.2 (KCNQ2; Q2) and K_v7.3 (KCNQ3; Q3) voltage‐dependent K⁺ channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug‐resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression‐burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named “KCNQ2 encephalopathy.” In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin‐1A, highlighting a novel pathogenetic mechanism for KCNQ2‐related epilepsies.     

The CC chemokine ligand 3 regulates CD11c+CD11b+CD8alpha- dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in T cell activation

The role of CC chemokine ligand 3 (CCL3) in activation of dendritic cells (DCs) following mouse hepatitis virus (MHV) infection of the central nervous system (CNS) was examined. The results indicate that CCL3 participates in an effective host response to MHV infection by contributing to CD11c+CD11b+CD8alpha- DC maturation, activation, and migration to cervical lymph nodes (CLN). Diminished CD8alpha- DC activation correlated with reduced IFN-gamma expression by virus-specific T cells accompanied by increased IL-10 production suggesting that CCL3 contributes to an effective host response to viral infection by enhancing the T cell activation potential of DC.     

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca²⁺ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK₈) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av) in a suspension of acinar cells. However, in the presence of 1.0 M Staurosporine the stimulatory effect of submaximal concentrations of CCK₈ was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca²⁺]_i,av in cells prestimulated with a submaximal concentration of CCK₈. The data obtained with staurosporine indicate that CCK₈-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca²⁺ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK₈-induced Ca²⁺ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK₈-evoked increase in [Ca²⁺]_i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK₈ at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca²⁺]_i,av evoked by the CCK₈ analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca²⁺]_i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK₈ concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca²⁺]_i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca²⁺ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca²⁺ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.