Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2.

The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 107 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-κB.

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling.

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

Phosphorylation of NF-kappaB p65 at Ser468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination.

The nuclear factor-kappaB (NF-kappaB) transcription factor system is a crucial component that controls several important biological functions, thus raising the need for mechanisms that ensure the correct termination of its activity. Here, we identify a new phosphorylation/ubiquitination switch in the NF-kappaB network that controls the stability of the transactivating p65 subunit. Tumour necrosis factor-induced phosphorylation of p65 at Ser468 allows binding of COMMD1 and cullin 2, components of a multimeric ubiquitin ligase complex mediating p65 ubiquitination. Mutation of p65 at Ser468 largely prevents p65 ubiquitination and proteasomal degradation. Inducible p65 elimination is restricted to a subset of NF-kappaB target genes such as Icam1. Accordingly, chromatin immunoprecipitation experiments reveal the selective recruitment of Ser468-phosphorylated p65 and COMMD1 to the Icam1 promoter. Phosphorylation of p65 at Ser468 leads to ubiquitin/proteasome-dependent removal of chromatin-bound p65, thus contributing to the selective termination of NF-kappaB-dependent gene expression.

NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation.

The serine/threonine kinase Cot triggers NF-kappaB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-kappaB-dependent gene expression. Also the C-terminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation.

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture.

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.

Influence of beta-adrenoceptor antagonists on hemorrhage-induced cellular immune suppression.

Hemorrhagic shock is associated with increasing catecholamine plasma concentrations. Plasma catecholamines are known to affect cellular immune functions. We therefore, investigated the effect of endogenously released catecholamines on lymphocyte distribution (CD4+ lymphocytes, CD8+ lymphocytes, and natural killer (NK) cells), splenocyte apoptosis (Annexin V binding), tumor necrosis factor-alpha (TNF-alpha), and interleukin 10 (IL-10) release during a volume-controlled hemorrhagic shock in mice. Mice received either saline (HEM), the non-selective beta-adrenoceptor antagonist propranolol (PROP; 2 mg/kg i.p.), or the beta1-adrenoceptor antagonist metoprolol (MET; 2 mg/kg i.p.) before induction of hemorrhage. Mice were sacrificed to obtain the spleen and whole blood 1 h after hemorrhage, 1 h after fluid resuscitation, and 24 h after hemorrhage. Flow cytometric analysis revealed an increase in circulating NK cells in the HEM group. This effect was completely abolished by pretreatment with propranolol or metoprolol. Furthermore, administration of either beta-adrenoceptor antagonist led to a decrease of circulating CD8+ lymphocyte numbers. Monitoring of splenocyte apoptosis by determination of Annexin V binding revealed an increase in splenocyte apoptosis 24 h after hemorrhage in the HEM group but not in the animals pretreated with propranolol or metoprolol. Induction of hemorrhage did not affect TNF-alpha or IL-10 plasma concentrations in either experimental group. We conclude that plasma catecholamines affect cellular immunity in the early phase of trauma via a beta-adrenergic pathway.

The effect of dehydroepiandrosterone on hemorrhage-induced suppression of cellular immune function.

OBJECTIVE: To determine whether the steroid hormone dehydroepiandrosterone (DHEA) improves cellular immune functions after hemorrhagic shock. DESIGN AND SETTING: Prospective controlled study in a research laboratory at an university medical center. SUBJECTS: Male NMRI mice. INTERVENTIONS: Animals received 0.9% saline or DHEA (20 mg/kg subcutaneously) before induction of a volume-controlled hemorrhagic shock (55% of estimated circulating blood volume) by retro-orbital puncture. One hour after hemorrhage mice underwent fluid resuscitation by intravenous infusion of lactated Ringer's solution (300% of the shed blood). Separate groups of mice were killed to obtain whole blood and spleen 1 h after hemorrhage, 1 h after fluid resuscitation, and 24 h after hemorrhage to determine lymphocyte distribution (CD4(+), CD8(+), NK1.1-AG(+)), splenocyte apoptosis, and plasma concentrations of tumor necrosis factor-alpha and interleukin-10. MEASUREMENTS AND RESULTS: Hemorrhage in control mice was associated with a rapid increase in circulating NK cell numbers. Elevated splenocyte apoptosis, an increased CD4/CD8 ratio, and decreased number of circulating CD8(+) T-cells was observed 24 h after hemorrhagic shock. DHEA administration was accompanied by a normalization of splenocyte apoptosis and lymphocyte migration. Induction of hemorrhagic shock did not affect TNF-alpha or IL-10 plasma concentrations in either treatment group. CONCLUSIONS: DHEA administration improves cellular immune function after hemorrhage and may therefore be beneficial in patients with hemorrhagic shock.