Treatment of inflammatory bowel disease associated E. coli with ciprofloxacin and E. coli Nissle in the streptomycin-treated mouse intestine

Background

E. coli belonging to the phylogenetic group B2 are linked to Inflammatory Bowel Disease (IBD). Studies have shown that antimicrobials have some effect in the treatment of IBD, and it has been demonstrated that E. coli Nissle has prophylactic abilities comparable to 5-aminosalicylic acid (5-ASA) therapy in ulcerative colitis. The objective of this study was to test if ciprofloxacin and/or E. coli Nissle could eradicate IBD associated E. coli in the streptomycin-treated mouse intestine.

Results

After successful colonization with the IBD associated E. coli strains in mice the introduction of E. coli Nissle did not result in eradication of either IBD associated strains or an E. coli from a healthy control, instead, co-colonization at high levels were obtained. Treatment of mice, precolonized with IBD associated E. coli, with ciprofloxacin for three days alone apparently resulted in effective eradication of tested E. coli. However, treatment of precolonized mice with a combination of ciprofloxacin for 3 days followed by E. coli Nissle surprisingly allowed one IBD associated E. coli to re-colonize the mouse intestine, but at a level 3 logs under E. coli Nissle. A prolonged treatment with ciprofloxacin for 7 days did not change this outcome.

Conclusions

In the mouse model E. coli Nissle can not be used alone to eradicate IBD associated E. coli; rather, 3 days of ciprofloxacin are apparently efficient in eradicating these strains, but surprisingly, after ciprofloxacin treatment (3 or 7 days), the introduction of E. coli Nissle may support re-colonization with IBD associated E. coli.     

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses

Background

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Methods

Replication‐defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7‐cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre‐terminal protein, and with a cre‐recombinase plasmid.

Results

We show that C7‐cre cells allow efficient production of gutted Ad using ΔE1 + ΔE2b + ΔE3 helper viruses whose growth can be limited by cre‐loxP‐mediated excision of the packaging signal. Gutted Ad vectors carrying ～28 kb cassettes expressing full‐length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%.

Conclusions

These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b‐deleted vectors display a profound reduction in viral gene expression. Copyright © 2002 John Wiley & Sons, Ltd.

     

New insight into HCV E1/E2 region of genotype 4a

Introduction

Hepatitis C virus (HCV) genome contains two envelope proteins (E1 and E2) responsible for the virus entry into the cell. There is a substantial lack of sequences covering the full length of E1/E2 region for genotype 4. Our study aims at providing new sequences as well as characterizing the genetic divergence of the E1/E2 region of HCV 4a using our new sequences along with all publicly available datasets.

Methods

The genomic segments covering the whole E1/E2 region were isolated from Egyptian HCV patients and sequenced. The resulting 36 sequences 36 were analyzed using sequence analysis techniques to study variability within and among hosts in the same time point. Furthermore, previously published HCV E1/E2 sequence datasets for genotype 4a were retrieved and categorized according to the geographical location and date of isolation and were used for further analysis of variability among Egyptian over a period of 15 years, also compared with non-Egyptian sequences to figure out region-specific variability.

Results

Phylogenetic analysis of the new sequences has shown variability within the host and among different individuals in the same time point. Analysis of the 36 sequences along with the Egyptian sequences (254 sequences in E1 in the period from 1997 to 2010 and 8 E2 sequences in the period from 2006 to 2010) has shown temporal change over time. Analysis of the new HCV sequences with the non-Egyptian sequences (182 sequences in E1 and 155 sequences in the E2) has shown region specific variability. The molecular clock rate of E1 was estimated to be 5E-3 per site per year for Egyptian and 5.38E-3 for non-Egyptian. The clock rate of E2 was estimated to be 8.48E per site per year for Egyptian and 6.3E-3 for non-Egyptian.

Conclusion

The results of this study support the high rate of evolution of the Egyptian HCV genotype 4a. It has also revealed significant level of genetic variability among sequences from different regions in the world.

     

Natural variation in the genes responsible for maturity loci E1, E2, E3 and E4 in soybean

Background and Aims

The timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering.

Methods

The sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1–E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded.

Key Results

A new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1–E3 loci was achieved using PCR-based markers. Allelic combinations at the E1–E4 loci were found to be associated with ecological types, and about 62–66 % of variation of flowering time could be attributed to these loci.

Conclusions

The study advances understanding of the combined roles of the E1–E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean,     

Apolipoprotein E phenotypes and hyperlipidemia

Summary Apolipoprotein E phenotypes were determined in 361 patients with hyperlipidemia and in controls. The E2 isoform was significantly more frequent in the group of hyperlipidemics (P<0.0005). This was not due to a higher frequency of E-2/2 homozygotes with type III hyperlipoproteinemia, but rather to a significantly higher frequency of E2 heterozygotes (P<0.0005). Subgrouping of hyperlipidemics into patients with a) hypertriglyceridemia, b) hypercholesterolemia and c) mixed hyperlipidemia revealed i) that isoform E2 was significantly more frequent in patients with hypertriglyceridemia (0.001>P>0.005), ii) that isoform E4 was significantly more frequent in patients with hypercholesterolemia (0.01>P>0.005) and iii) that isoforms E2 (P>0.005) and E4 (0.05>P>0.025) were both more frequent in patients with mixed hyperlipidemia. Roughly 20% of patients with mixed hyperlipidemia had one of the rare phenotypes E-4/4,-4/2 or-2/2. We conclude that alleles 2 and 4 both contribute to the susceptibility for, and/or phenotypic expression of hyperlipidemia. Whereas the gene 2 seems to exert its influence on plasma lipoproteins by an abnormal gene product (E2) that has reduced binding activity to lipoprotein receptors, the mechanism underlying the association of the 4 gene with hyperlipidemia is presently unclear.     

High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli

Background

Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.

Methodology/Principal Findings

E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m²) combination with different temperature (22, 28, 30, 32, and 37°C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.

Conclusions/Significance

These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.     

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

Background

Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2^c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.

Methods

We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.

Findings

Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.

Conclusions

The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.     

Comparison of M.I.C.E. and Etest with CLSI Agar Dilution for Antimicrobial Susceptibility Testing against Oxacillin-Resistant Staphylococcus spp

Objective

The main objective of this study was to comparatively evaluate the performance of M.I.C.E. and Etest methodologies to that of agar dilution for determining the antimicrobial susceptibility profile of oxacillin-resistant Staphylococcus spp.

Methods

A total of 100 oxacillin-resistant Staphylococcus spp. isolates were collected from hospitalized patients at a teaching hospital. Antimicrobial susceptibility testing for vancomycin, teicoplanin and linezolid was performed using the reference CLSI agar dilution method (2009), Etest and M.I.C.E. methodologies. The MIC values were interpreted according to CLSI susceptibility breakpoints and compared by regression analysis.

Results

In general, the essential agreement (±1-log₂) between M.I.C.E. and CLSI agar dilution was 93.0%, 84.0% and 77.0% for linezolid, teicoplanin and vancomycin, respectively. Essential agreement rates between M.I.C.E. and Etest were excellent (>90.0%) for all antibiotics tested. Both strips (M.I.C.E. and Etest) yielded two very major errors for linezolid. Unacceptable minor rates were observed for teicoplanin against CoNS and for vancomycin against S. aureus.

Conclusions

According to our results, linezolid and teicoplanin MICs against all staphylococci and S. aureus, respectively, were more accurately predicted by M.I.C.E. strips. However, the Etest showed better performance than M.I.C.E. for predicting vancomycin MICs against all staphylococci. Thus, microbiologists must be aware of the different performance of commercially available gradient strips against staphylococci.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

Dihaploids of Elymus from the interspecific crosses E. dolichatherus x E. tibeticus and E. brevipes x E. panormitanus

Summary Dihaploids (n=2x=14, SY) of two Elymus species, i.e., E. dolichatherus (Keng) Löve (2n=4x=28, SSYY) and E. brevipes (Keng) Löve (2n=4x=28, SSYY), were obtained from the interspecific hybrid combinations E. dolichatherus () x E. tibeticus (Meld.) G. Singh () and E. brevipes () x E. panormitanus (Parl.) Tzvelev (). The dihaploids were probably formed through selective elimination of male parental chromosomes in early embryo development. Meiotic chromosome behavior was studied in E. dolichatherus, E. brevipes, and their dihaploids. The two parental Elymus species had regular meioses with predominantly ring bivalent formation. A low frequency of homoeologous chromosome pairing was observed, with an average of 0.81 bivalents and 0.03 trivalents in the dihaploid of E. dolichatherus, and 0.26 bivalents in the dihaploid of E. brevipes. Up to two chromatid bridges accompanied by small fragments were present at anaphase I of the E. dolichatherus dihaploid. It is concluded from this study that: (i) both E. dolichatherus and E. brevipes are allotetraploid species; (ii) a low affinity exists between the S and Y genomes of the two Elymus species.     

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Background

The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.

Methodology/Principal Findings

In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.

Conclusions/Significance

A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.     

Microarray-based analysis and clinical validation identify ubiquitin-conjugating enzyme E2E1 (<Emphasis Type="Italic">UBE2E1</Emphasis>) as a prognostic factor in acute myeloid leukemia

Background

Previous research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions.

Methods

We performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis.

Results

Microarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p?<?0.05). Next, we validated our findings in an independent cohort of AML samples collected in our hospital. We found that ubiquitin-conjugating enzyme E2E1 (UBE2E1) expression was adversely correlated with AML survival (p?=?0.04). Multivariable analysis showed that UBE2E1 ^high patients had a significant shorter OS and shorter progression-free survival after adjusting other known prognostic factors (p?=?0.03). At last, we found that UBE2E1 expression was negatively correlated with patients’ response to induction chemotherapy (p?<?0.05).

Conclusions

In summary, we demonstrated that UBE2E1 expression was a novel prognostic factor in adult, non-APL AML patients.

     

Selective detection of Cathepsin E proteolytic activity

Background

Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide–substrate that is only sensitive to Cath E but not Cath D.

Methods

Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH₂, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence.

Results

Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K_cat/K_M) of Cath E for substrate e was 16.7 μM⁻¹ S⁻¹. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B.

Conclusions

This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement.

General significance

This study forms the foundation of Cath E specific inhibitor development in further studies.     

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector

Objective

To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.

Results

The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.

Conclusions

The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

Background

Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.

Methods

Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.

Results

The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.

Conclusion

Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.