Trans-acting genetic variants have a substantial, albeit poorly characterized, role in the heritable determination of gene expression. Using paired purified primary monocytes and B cells, we identify new predominantly cell type-specific cis and trans expression quantitative trait loci (eQTLs), including multi-locus trans associations to LYZ and KLF4 in monocytes and B cells, respectively. Additionally, we observe a B cell-specific trans association of rs11171739 at 12q13.2, a known autoimmune disease locus, with IP6K2 (P = 5.8 × 10(-15)), PRIC285 (P = 3.0 × 10(-10)) and an upstream region of CDKN1A (P = 2 × 10(-52)), suggesting roles for cell cycle regulation and peroxisome proliferator-activated receptor γ (PPARγ) signaling in autoimmune pathogenesis. We also find that specific human leukocyte antigen (HLA) alleles form trans associations with the expression of AOAH and ARHGAP24 in monocytes but not in B cells. In summary, we show that mapping gene expression in defined primary cell populations identifies new cell type-specific trans-regulated networks and provides insights into the genetic basis of disease susceptibility.

OBJECTIVE

Reduced ileal Paneth cell alpha-defensin expression has been reported to be associated with Crohn's disease, especially in patients carrying NOD2 mutations. The aim of this study was to independently assess whether NOD2, alpha-defensins and Crohn's disease are linked.

METHODS

Using quantitative real-time polymerase chain reaction (RT-PCR), we measured the mRNA expression levels of key Paneth cell antimicrobial peptides (DEFA5, DEFA6, LYZ, PLA2G2A), inflammatory cytokines [interkelukin 6 (IL6) and IL8], and a marker of epithelial cell content, villin (VIL1) in 106 samples from both affected ileum (inflamed Crohn's disease cases, n = 44) and unaffected ileum (non-inflamed; Crohn's disease cases, n = 51 and controls, n = 11). Anti-human defensin 5 (HD-5) and haematoxylin/eosin immunohistochemical staining was performed on parallel sections from NOD2 wild-type and NOD2 mutant ileal Crohn's disease tissue.

RESULTS

In Crohn's disease patients, DEFA5 and DEFA6 mRNA expression levels were 1.9- and 2.2-fold lower, respectively, in histologically confirmed inflamed ileal mucosa after adjustment for confounders (DEFA5, p<0.001; DEFA6, p = 0.001). In contrast to previous studies, we found no significant association between alpha-defensin expression and NOD2 genotype. HD-5 protein data supports these RNA findings. The reduction in HD-5 protein expression appears due to surface epithelial cell loss and reduced Paneth cell numbers as a consequence of tissue damage.

CONCLUSIONS

Reduction in alpha-defensin expression is independent of NOD2 status and is due to loss of surface epithelium as a consequence of inflammatory changes rather than being the inciting event prior to inflammation in ileal Crohn's disease.

OBJECTIVE

Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1(+) versus PD-L1(-) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1(+) melanoma microenvironment coordinately contributing to immunosuppression.

METHODS

Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1(+) versus PD-L1(-) melanomas.

RESULTS

Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1(+) melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1(+) melanomas, involved in CD8(+) T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells.

CONCLUSIONS

These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function. Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane. Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endolysin to the membrane but also for its release into the periplasm. The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

OBJECTIVE

Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are precursors to gastric carcinogenesis. We sought to identify molecular biomarkers of gastric metaplasias and gastric cancer by gene expression profiling of metaplastic lesions from patients.

METHODS

Complementary DNA microarray analysis was performed on IM and SPEM cells isolated from patient samples using laser capture microdissection. Up-regulated transcripts in metaplastic lesions were confirmed by immunostaining analysis in IM, SPEM, and gastric cancer tissues. Proteins that were highly expressed specifically in gastric cancer tissues were analyzed for their association with survival in a test set (n = 450) and a validation set (n = 502) of samples from gastric cancer patients.

RESULTS

Compared with normal chief cells, 858 genes were differentially expressed in IM or SPEM samples. Immunostaining was detected for 12 proteins, including 3 new markers of IM (ACE2, LGALS4, AKR1B10) and 3 of SPEM (OLFM4, LYZ, DPCR1). Of 13 proteins expressed in IM or SPEM, 8 were expressed by 17%-50% of human gastric cancer tissues (MUC13, OLFM4, CDH17, KRT20, MUC5AC, LGALS4, AKR1B10, REG4). Expression of CDH17 or MUC13 correlated with patient survival in the test and validation sets. Multivariate analysis showed that CDH17 was an independent prognostic factor in patients with stage I or node-negative disease.

CONCLUSIONS

We identified several novel biomarkers for IM, SPEM, and gastric cancer using gene expression profiling of human metaplastic lesions. Expression of CDH17 and MUC13 was up-regulated in gastric cancer tissues. CDH17 is a promising prognostic marker for early stage gastric cancer.

The P1 lysozyme Lyz is secreted to the periplasm of Escherichia coli and accumulates in an inactive membrane-tethered form. Genetic and biochemical experiments show that, when released from the bilayer, Lyz is activated by an intramolecular thiol-disulfide isomerization, which requires a cysteine in its N-terminal SAR (signal-arrest-release) domain. Crystal structures confirm the alternative disulfide linkages in the two forms of Lyz and reveal dramatic conformational differences in the catalytic domain. Thus, the exported P1 endolysin is kept inactive by three levels of control-topological, conformational, and covalent-until its release from the membrane is triggered by the P1 holin.

We conducted a genome-wide association study to identify novel genes influencing diastolic blood pressure (BP) response to hydrochlorothiazide, a commonly prescribed thiazide diuretic preferred for the treatment of high BP. Affymetrix GeneChip Human Mapping 100K Arrays were used to measure single nucleotide polymorphisms across the 22 autosomes in 194 non-Hispanic black subjects and 195 non-Hispanic white subjects with essential hypertension selected from opposite tertiles of the race- and sex-specific distributions of age-adjusted diastolic BP response to hydrochlorothiazide (25 mg daily, PO, for 4 weeks). The black sample consisted of 97 "good" responders (diastolic BP response [mean+/-SD]=-18.3+/-4.2 mm Hg; age=47.1+/-6.1 years; 51.5% women) and 97 "poor" responders (diastolic BP response=-0.18+/-4.3; age=47.4+/-6.5 years; 51.5% women). Haplotype trend regression identified a region of chromosome 12q15 in which haplotypes constructed from 3 successive single nucleotide polymorphisms (rs317689, rs315135, and rs7297610) in proximity to lysozyme (LYZ), YEATS domain containing 4 (YEATS4), and fibroblast growth receptor substrate 2 (FRS2) were significantly associated with diastolic BP response (nominal P=2.39 x 10(-7); Bonferroni corrected P=0.024; simulated experiment-wise P=0.040). Genotyping of 35 additional single nucleotide polymorphisms selected to "tag" linkage disequilibrium blocks in these genes provided corroboration that variation in LYZ and YEATS4 was associated with diastolic BP response in a statistically independent data set of 291 black subjects and in the sample of 294 white subjects. These results support the use of genome-wide association analyses to identify novel genes influencing antihypertensive drug responses.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of alpha-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu(-/-) and Naglu(+/-) mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu(-/-) mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies-a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

Ischemia-reperfusion (I/R) injury is a process whereby an initial hypoxic insult and subsequent return of blood flow leads to the propagation of innate immune responses and organ injury. The necessity of the pattern recognition receptor, Toll-like receptor (TLR)4, for this innate immune response has been previously shown. However, TLR4 is present on various cell types of the liver, both immune and nonimmune cells. Therefore, we sought to determine the role of TLR4 in individual cell populations, specifically, parenchymal hepatocytes (HCs), myeloid cells, including Kupffer cells, and dendritic cells (DCs) subsequent to hepatic I/R. When HC-specific (Alb-TLR4(-/-) ) and myeloid-cell-specific (Lyz-TLR4(-/-) ) TLR4 knockout (KO) mice were subjected to warm hepatic ischemia, there was significant protection in these mice, compared to wild type (WT). However, the protection afforded in these two strains was significantly less than global TLR4 KO (TLR4(-/-) ) mice. DC-specific TLR4(-/-) (CD11c-TLR4(-/-) ) mice had significantly increased hepatocellular damage, compared to WT mice. Circulating levels of high-mobility group box 1 (HMGB1) were significantly reduced in Alb-TLR4(-/-) mice, compared to WT, Lyz-TLR4(-/-) , CD11c-TLR4(-/-) mice and equivalent to global TLR4(-/-) mice, suggesting that TLR4-mediated HMGB1 release from HCs may be a source of HMGB1 after I/R. HCs exposed to hypoxia responded by rapidly phosphorylating the mitogen-activated protein kinases, c-Jun-N-terminal kinase (JNK) and p38, in a TLR4-dependent manner; inhibition of JNK decreased release of HMGB1 after both hypoxia in vitro and I/R in vivo.

CONCLUSIONS

These results provide insight into the individual cellular response of TLR4. The parenchymal HC is an active participant in sterile inflammatory response after I/R through TLR4-mediated activation of proinflammatory signaling and release of danger signals, such as HMGB1.

BACKGROUND

Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (IMI) with S. uberis and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with S. uberis O140J.

RESULTS

Streptococcus uberis IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (DEG). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were signaling pathways. Among the most inhibited were LXR/RXR Signaling and PPARalpha/RXRalpha Signaling. Pathways activated by IMI were IL-10 Signaling and IL-6 Signaling which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA3. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Network analysis of DEG indicated that TNF had positive relationships with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1).

CONCLUSIONS

Results provided novel information into the early signaling and metabolic pathways in mammary tissue that are associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to potent activation of pro-inflammatory pathways that were associated with a marked inhibition of lipid synthesis, stress-activated kinase signaling cascades, and PPAR signaling (most likely PPARgamma). This latter effect may provide a mechanistic explanation for the inverse relationship between immune response and milk fat synthesis.

BACKGROUND

Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration.

METHODS

Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature.

RESULTS

Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen.

CONCLUSIONS

The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.

Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.

The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell α-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI>> 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human α-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise.

Many biomedical applications of gold nanoparticles (NPs) rely on proteins that are covalently attached or adsorbed on the NP surface. The biological functionality of the protein-NP conjugate depends on the protein's ability to interact with target molecules, which is affected by NP characteristics such as size, curvature, aspect ratio, morphology, crystal structure, and surface chemistry. In the present study, the effect of gold nanoparticle morphology on the structure and function of adsorbed enzymes, lysozyme (Lyz) and α-chymotrypsin (ChT), has been investigated. Gold nanospheres (AuNS) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm. Under saturating conditions, proteins adsorb with a higher surface density on AuNR when compared to AuNS. In the case of Lyz, adsorption on AuNS and AuNR resulted in a 10% and 15% loss of secondary structure, respectively, leading to conjugate aggregation and greatly reduced enzymatic activity. ChT retained most of its secondary structure and activity on AuNS and AuNR at low surface coverages; however, as protein loading approached monolayer conditions on AuNR, a 40% loss in secondary structure and 86% loss of activity was observed. Subsequent adsorption of ChT in multilayers on the AuNR surface allowed the conjugates to recover activity and remain stable. It is clear that AuNP morphology does affect adsorbed protein structure; a better understanding of these differences will be essential to engineer fully functional nanobioconjugates.

The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14-1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.

MicroRNA (miRNA) are small single-stranded noncoding RNA with important roles in regulating innate immunity in nonruminants via transcriptional and posttranscriptional mechanisms. Mastitis causes significant losses in the dairy industry and a wealth of large-scale mRNA expression data from mammary tissue have provided fundamental insights into the tissue adaptations to pathogens. We studied the expression of 14 miRNA (miR-10a, -15b, -16a, -17, -21, -31, -145, -146a, -146b, -155, -181a, -205, -221, and -223) associated with regulation of innate immunity and mammary epithelial cell function in tissue challenged with Streptococcus uberis. Those data, along with microarray expression of 2,102 differentially expressed genes, were used for bioinformatics analysis to uncover putative target genes and the most affected biological pathways and functions. Three miRNA (181a, 16, and 31) were downregulated approximately 3- to 5-fold and miR-223 was upregulated approximately 2.5-fold in infected versus healthy tissue. Among differentially expressed genes due to infection, bioinformatics analysis revealed that the studied miRNA share in the regulation of a large number of metabolic (SCD, CD36, GPAM, and FASN), immune/oxidative stress (TNF, IL6, IL10, SOD2, LYZ, and TLR4), and cellular proliferation/differentiation (FOS and CASP4) target genes. This level of complex regulation was underscored by the coordinate effect revealed by bioinformatics on various cellular pathways within the Kyoto Encyclopedia of Genes and Genomes database. Most pathways associated with "cellular processes," "organismal systems," and "diseases" were activated by putative target genes of miR-31 and miR-16a, with an overlapping activation of "immune system" and "signal transduction." A pronounced effect and activation of miR-31 target genes was observed within "folding, sorting, and degradation," "cell growth and death," and "cell communication" pathways, whereas a marked inhibition of "lipid metabolism" occurred. Putative targets of miR-181a had a strong effect on FcγR-mediated phagocytosis, toll-like receptor signaling, and antigen processing and presentation, which were activated during intramammary infections. The targets of both miR-31 and miR-223 had an inhibitory effect on "lipid metabolism." Overall, the combined analyses indicated that changes in mammary tissue immune, metabolic, and cell growth-related signaling pathways during infection might have been mediated in part through effects of miRNA on gene transcription. Differential expression of miRNA supports the view from nonruminant cells/tissues that certain miRNA might be essential for the tissue's adaptive response to infection.

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N(2) as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water-acetic acid for folded cyt c and Lyz. Thus, protein CSD from water-acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.

Recently, we reported development of the C57BL/6.NOD-Aec1Aec2 mouse carrying two genetic intervals derived from the NOD mouse. These two genetic regions confer Sjögren's syndrome (SjS)-like disease in SjS-non-susceptible C57BL/6 mice. In an attempt to define the molecular bases underlying onset of dacryoadenitis and subsequently keratoconjunctivitis sicca (or xerophthalmia) in the C57BL/6.NOD-Aec1Aec2 mouse model, we have carried out a study utilizing microarray technology. Using oligonucleotide microarrays, gene expression profiles of lacrimal glands at 4, 8, 12, 16 and 20weeks of age were generated for C57BL/6.NOD-Aec1Aec2 male mice. Analyses using Linear Models for Microarray Analysis package and B-statistics, 552 genes were identified as being differentially expressed (adjusted p-value <0.01 and B <1.5) during the development of SjS-like disease. These 552 genes could be arranged into four clusters, with each cluster defining a unique pattern of temporal expression, while the individual genes within each cluster could be grouped according to related function. Using a pair-wise analysis, temporal changes in gene expressions provided profiles indicating that individual genes were differentially expressed at specific time points during development of SjS. In addition, multiple genes that have been reported to show, either in humans or mouse models, an association with autoimmunity and/or SjS, e.g., ApoE, Baff, Clu, Ctla4, Fas/Fasl, Irf5, Lyzs, Nfkb, Socs3, Stat4, Tap2, Tgfbeta1, Tnfa, and Vcam1 were also found to exhibit differential expressions, both quantitatively and temporally. Selecting a few families of genes, e.g., cystatins, cathepsins, metalloproteinases, lipocalins, complement, kallikreins, carbonic anhydrases and tumor necrosis factors, it was noted that only a limited number of family members showed differential expressions, suggesting a restricted glandular expression. Utilizing these genes, pathways of inter-reactive genes have been constructed for apoptosis and fatty acid homeostasis, leading to modeling of possible underlying events inducing disease. Thus, these different approaches to analyze microarray data permit identification of multiple sets of genes of interest whose expressions and expression profiles may correlate with molecular mechanisms, signaling pathways and/or immunological processes involved in the development and onset of SjS in this mouse model, thereby providing new insight into the underlying cause or regulation of this disease.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

Olfactory ensheathing cells (OECs) accompany the axons of olfactory receptor neurons, which regenerate throughout life, from the olfactory mucosa into the olfactory bulb. OECs have shown widely varying efficacy in repairing the injured nervous system. Analysis of the transcriptome of OECs will help in understanding their biology and will provide tools for investigating the mechanisms of their efficacy and interactions with host tissues in lesion models. In this study, we compared the transcriptional profile of cultured OECs with that of Schwann cells (SCs) and astrocytes (ACs), two glial cell types to which OECs have similarities. Two biological replicates of RNA from cultured OECs, SCs, and ACs were hybridized to long oligo rat 5K arrays against a common reference pool of RNA (50% cultured fibroblast RNA and 50% neonatal rat brain RNA). Transcriptional profiles were analyzed by hierarchical clustering, Principal Components Analysis, and the Venn diagram. The three glial cell types had similarly increased or decreased expression of numerous transcripts compared with the reference. However, OECs were distinguishable from both SCs and ACs by a modest number of transcripts, which were significantly enriched or depleted. Furthermore, OECs and SCs were more closely related to each other than to ACs. Expression of selected transcripts not previously characterized in OECs, such as Lyz, Timp2, Gro1 (Cxcl1), Ccl2 (MCP1), Ctgf, and Cebpb, was validated by real-time reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry in cultured OECs, SCs, and ACs, and adult tissues was performed to demonstrate their expression at the protein level.

Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr(tm1UNC)) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4-day-old CF and wild-type (WT) mice and increased similarly in both groups of mice between 12 and 16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared with WT, whereas Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice, which is before Paneth cell development. With the use of gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared with WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice but did significantly slow intestinal transit in WT mice, suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice, and this is likely a major factor in SIBO in CF.

NUP98-HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment-related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98-HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98-HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1 expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98-HOXA9. Following irradiation, NUP98-HOXA9-expressing embryos showed increased numbers of cells in G2-M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98-HOXA9-induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98-HOXA9-transgenic fish developed a myeloproliferative neoplasm (MPN) at 19-23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high-throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.