C/EBPß-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation.

The transcription factor C/EBPß plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPß isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPß in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation.

Altered NK cell function in obese healthy humans.

BACKGROUND: Obesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (<25 kg/m(2)) and obese (>30 kg/m(2)). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme(+) and IFN-γ(+) NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined. RESULTS: As opposed to similar NK cell counts, the number of CD3(+)CD56(+) cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells. CONCLUSIONS: NK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge.

Regulation of monocyte differentiation by specific signaling modules and associated transcription factor networks.

Monocyte/macrophages are important players in orchestrating the immune response as well as connecting innate and adaptive immunity. Myelopoiesis and monopoiesis are characterized by the interplay between expansion of stem/progenitor cells and progression towards further developed (myelo)monocytic phenotypes. In response to a variety of differentiation-inducing stimuli, various prominent signaling pathways are activated. Subsequently, specific transcription factors are induced, regulating cell proliferation and maturation. This review article focuses on the integration of signaling modules and transcriptional networks involved in the determination of monocytic differentiation.

Cotrimoxazole plasma levels, dialyzer clearance and total removal by extended dialysis in a patient with acute kidney injury: risk of under-dosing using current dosing recommendations.

BACKGROUND: Dosing of antibiotics in critically ill patients is challenging. It becomes even more difficult if renal or hepatic impairment ensue. Modern means of renal replacement therapy are capable of removing antibiotics to a higher rate than decades ago, leaving clinicians with a high degree of uncertainty concerning the dose of antibiotics in this patient population. Cotrimoxazole, a combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is frequently used in the treatment of several infections including Pneumocystis jirovecii pneumonia (PCP). CASE PRESENTATION: Here we describe a patient with acute kidney injury in which we investigated the TMP and SMX levels during the course of an ICU stay. Cotrimoxazole was administered every six hours i.v. in a dose of TMP/SMX 15/75 mg/kg/day. Extended dialysis was performed with a high-flux dialyzer. Blood samples, as well as pre- and postdialyzer samples and aliquots of the collected spent dialysate were collected.Observed peak concentrations (Cmax) were 7.51 mg/l for TMP and 80.80 mg/l for SMX. Decline of blood levels during extended dialysis (TMP 64%; SMX 84%) was mainly due to removal by the dialysis procedure, illustrated by the high dialyzer clearances (median of 4 extended dialysis sessions: TMP 94.0 / SMX 51.0 ml/min), as well as by the absolute amount of both substances in the collected spent dialysate (median of 6 extended dialysis sessions: TMP 556 mg / SMX 130 mg). Within the limitation of a case report our data from 4 consecutive extended dialysis sessions suggest that this procedure substantially removes both TMP and SMX. CONCLUSIONS: Dose reduction, which is usually advocated in patients with acute kidney injury under renal replacement therapy, might lead to significant under-dosing. Pharmacokinetic studies for TMP/SMX dosing in this patient population are necessary to allow adequate dosing.

The Enhanced Liver Fibrosis (ELF) score: normal values, influence factors and proposed cut-off values.

BACKGROUND & AIMS: Progressive fibrosis is a major cause of morbidity and mortality in chronic liver disease. To replace liver biopsy for disease staging, multiple serum markers are under evaluation with multiparametric panels yielding the most promising results. The Enhanced Liver Fibrosis (ELF) score is an ECM marker set consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) showing good correlations with fibrosis stages in chronic liver disease. METHODS: The ELF score was measured in 400 healthy controls and 79 chronic hepatitis C patients using an ADVIA Centaur automated system. The ELF score was calculated using the published algorithm combining TIMP-1, PIIINP and HA values. Patients' fibrosis stage was defined histologically. ROC analyses were performed to study marker validity. Reference values and influence factors for the ELF score were validated. RESULTS: ELF score reference values ranged from 6.7 to 9.8 and were significantly higher for men vs. women (7.0-9.9 vs. 6.6-9.3, respectively). Afternoon values were slightly higher than morning values (6.7-9.9 vs. 6.6-9.5, respectively). Age was a notable influence factor. We identified three cut-off values: 7.7 for a high sensitivity exclusion of fibrosis, 9.8 for a high specificity identification of fibrosis (sensitivity 69%, specificity 98% for moderate fibrosis), and 11.3 to discriminate cirrhosis (sensitivity 83%, specificity 97%). ELF score validity was superior to the results of the single tests. CONCLUSIONS: The ELF score can predict moderate fibrosis and cirrhosis. However, influence factors such as gender and age need to be taken into account.

Regulation of C/EBPß and resulting functions in cells of the monocytic lineage.

Monocyte/macrophages play an important role in orchestrating the immune response. The present review refers to C/EBPß, which is a key transcription factor regulating monocytic gene expression. Following a general introduction to C/EBPß, this article focuses on activators and regulators of the C/EBPß system in monocytic cells, including differentiating agents, cytokines, and bacterial products as well as associated signaling pathways. Furthermore, C/EBPß target genes in monocytic cells are summarized and resulting functions are described, including regulation of proliferation and differentiation as well as orchestration of processes of mainly the innate immune response. In addition, a variety of disease stages are described in which a dysregulation of the C/EBPß system may be involved. A detailed knowledge of the C/EBPß system in monocytic cells may help to further understand the difference between inflammatory and malignant proliferation as well as additional regulatory facets of innate immunity.

New insights into the function of the iron deficiency-induced protein C from Synechococcus elongatus PCC 7942.

Iron limitation has a strong impact on electron transport reactions of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942 (thereafter referred to as S. elongatus). Among the various adaptational processes on different cellular levels, iron limitation induces a strongly enhanced expression of IdiC (iron-deficiency-induced protein C). In this article, we show that IdiC is loosely attached to the thylakoid and to the cytoplasmic membranes and that its expression is enhanced during conditions of iron starvation and during the late growth phase. The intracellular IdiC level was even more increased when additional iron was replenished in the late growth phase. On the basis of its amino acid sequence and of its absorbance spectrum, IdiC can be classified as a member of the family of thioredoxin (TRX)-like (2Fe-2S) ferredoxins. The presence of an iron cofactor in IdiC was detected by inductive coupled plasma optical emission spectrometry (ICP-OES). Comparative measurements of electron transport activities of S. elongatus wild type (WT) and an IdiC-merodiploid mutant called MuD, which contained a strongly reduced IdiC content under iron-sufficient as well as iron-deficient growth conditions, were performed. The results revealed that MuD had a strongly increased light sensitivity, especially under iron limitation. The measurements of photosystem II (PS II)-mediated electron transport rates in WT and MuD strain showed that PS II activity was significantly lower in MuD than in the WT strain. Moreover, P(700) (+) re-reduction rates provided evidence that the respiratory activities, which were very low in the MuD strain in the presence of iron, significantly increased in iron-starved cells. Thus, an increase in respiration may compensate for the drastic decrease of photosynthetic electron transport activity in MuD grown under iron starvation. Based on the similarity of the S. elongatus IdiC to the NuoE subunit of the NDH-1 complex in Escherichia coli, it is likely that IdiC has a function in the electron transport processes from NAD(P)H to the plastoquinone pool. This is in agreement with the up-regulation of IdiC in the late growth phase as well as under stress conditions when PS II is damaged. As absence or high reduction of the IdiC level would prevent or reduce the formation of functional NDH-1 complexes, under such conditions electron transport routes via alternative substrate dehydrogenases, donating electrons to the plastoquinone pool, can be assumed to be up-regulated.

CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology.

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein ß (C/EBPß). Initially, we demonstrated a marked increase in nuclear C/EBPß-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPß effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPß) cell lines stably overexpressing C/EBPß isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPß-long), but not those overexpressing LIP (C/EBPß-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPß-short cells. In C/EBPß(WT) macrophage-like cells (high endogenous C/EBPß), we measured a reduced proliferation/cycling index compared with C/EBPß(KO). The typical macrophage morphology was only observed in C/EBPß(WT), whereas C/EBPß(KO) stayed round. C/EBPα did not compensate for C/EBPß effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPß(KO) macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPß-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPß-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPß-long and C/EBPß(WT) cells exhibited low E2F1 and cyclin E levels, and C/EBPß overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPß reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPß may be important for coordinated monocytic differentiation.

Evaluation of an automated capillary electrophoresis system for serum protein electrophoresis with the determination of gender-specific reference values.

In this report we summarize evaluation data of an automated capillary zone electrophoresis (Capillarys II, Sebia, France) in comparison to a conventional cellulose acetate electrophoresis system (Hite320, Olympus, Germany). Serum samples from 428 blood donors were analysed to evaluate gender-specific reference intervals, which revealed significant percentage differences between male and female. In contrast to conventional electrophoresis methods all lipoproteins co-migrate with the albumin fraction using capillary electrophoresis. Comparisons between both separation methods and quantitative determinations of albumin/total protein show a better correlation between the albumin fraction determined by capillary electrophoresis and the quantitative determinations. A transversal evaluation of serum protein fractions should be performed within one day due to the combination of effects based on serum sample instability and method imprecision.

Transcript profiling reveals new insights into the acclimation of the mesophilic fresh-water cyanobacterium Synechococcus elongatus PCC 7942 to iron starvation.

The regulatory network for acclimation of the obligate photoautotrophic fresh water cyanobacterium Synechococcus elongatus PCC 7942 to iron (Fe) limitation was studied by transcript profiling with an oligonucleotide whole genome DNA microarray. Six regions on the chromosome with several Fe-regulated genes each were identified. The irpAB and fut region encode putative Fe uptake systems, the suf region participates in [Fe-sulfur] cluster assembly under oxidative stress and Fe limitation, the isiAB region encodes CP43' and flavodoxin, the idiCB region encodes the NuoE-like electron transport associated protein IdiC and the transcriptional activator IdiB, and the ackA/pgam region encodes an acetate kinase and a phosphoglycerate mutase. We also investigated the response of two S. elongatus PCC 7942 mutants to Fe starvation. These were mutant K10, lacking IdiB but containing IdiC, and mutant MuD, representing a idiC-merodiploid mutant with a strongly reduced amount of IdiC as well as IdiB. The absence of IdiB in mutant K10 or the strongly reduced amount of IdiB in mutant MuD allowed for the identification of additional members of the Fe-responsive IdiB regulon. Besides idiA and the irpAB operon somB(1), somA(2), ftr1, ackA, pgam, and nat also seem to be regulated by IdiB. In addition to the reduced amount of IdiB in MuD, the low concentration of IdiC may be responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to the wild type and mutant K10. This fact may explain why it has been impossible to obtain a fully segregated IdiC-free mutant, whereas it was possible to obtain a fully segregated IdiB-free mutant.

Characterization of the putative iron sulfur protein IdiC (ORF5) in Synechococcus elongatus PCC 7942.

The IdiC protein (iron deficiency induced protein C) is encoded by orf5 (now called idiC), which is part of the iron-responsive idiB operon of Synechococcus elongatus PCC 7942. The 20.5 kDa IdiC protein has a putative transmembrane helix and belongs to the thioredoxin (TRX)-like [2Fe-2S] ferredoxin family. IdiC has the highest similarity to the peripheral subunit NuoE of the Escherichia coli NDH-1 complex. IdiC expression increased under iron starvation and also in the late growth phase, representing growth conditions, which favor photosynthetic cyclic and respiratory electron transport over photosynthetic linear electron transport from water to NADP+. Attempts to insertionally inactivate the idiC gene generated merodiploid mutants with a strongly reduced IdiC content (mutant MuD) but no IdiC-free mutant. Thus, IdiC seems to be an essential protein for the viability of S. elongatus under the used experimental conditions. Comparative analyses of S. elongatus wild type (WT) and mutant MuD showed that under iron limitation in WT and MuD the amount of the reaction center proteins PsbA and PsaA/B was highly reduced. MuD had a lower growth rate, chlorophyll content, and photosynthetic O2 evolving activity with bicarbonate as electron acceptor than WT. Immunoblot analyses also showed that in MuD, when grown under iron limitation, the amount of the proteins IdiC and IdiB was greatly reduced as compared to WT. As a consequence of the reduction of the transcription factor IdiB, IdiA and IrpA expression were also decreased. In addition, the IsiA protein concentration was lower in MuD than in WT, although the isiA mRNA was equally high in MuD and WT. Another significant difference was the lower expression of the ferredoxin:NADP+ oxidoreductase in mutant MuD under iron limitation compared to WT. A possible function of the protein IdiC in cyclic electron transport around photosystem I and/or in respiratory electron transport will be discussed.