Bruton's tyrosine kinase is required for apoptotic cell uptake via regulating the phosphorylation and localization of calreticulin.

In addition to regulating B cell development and activation, Bruton's tyrosine kinase (Btk) functions downstream of multiple TLRs, including TLR7, to regulate innate immune responses in myeloid cells. Although critical for defense against RNA viruses such as influenza and Sendai virus, recognition of self-RNA by TLR7 also has been shown to be an important contributor to the pathophysiology of systemic lupus erythematosus. To date, the role of Btk in regulating TLR7-mediated responses is poorly understood. In the current study, we have demonstrated a hitherto undiscovered role for Btk in apoptotic cell uptake, identifying the molecular chaperone calreticulin (CRT) as a novel substrate for Btk in regulating this response. CRT together with the transmembrane receptor CD91 function at the cell membrane and regulate uptake of C1q-opsonised apoptotic cells. Our results show that Btk directly phosphorylates CRT and that in the absence of Btk, CRT fails to localize with CD91 at the cell surface and at the phagocytic cup. Critically, a blocking Ab against CRT in wild-type macrophages mimics the inability of Btk-deficient macrophages to phagocytose apoptotic cells efficiently, indicating the critical importance of Btk in regulating CRT-driven apoptotic cell uptake. Our data have revealed a novel regulatory role for Btk in mediating apoptotic cell clearance, with CRT identified as the critical component of the CRT/CD91/C1q system targeted by Btk. Given the importance of clearing apoptotic cell debris to prevent inappropriate exposure of TLRs to endogenous ligands, our results have important implications regarding the role of Btk in myeloid cell function.

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-κB (NF-κB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-κB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-κB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-κB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-κB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-κB activity in neurons.

Defects in acute responses to TLR4 in Btk-deficient mice result in impaired dendritic cell-induced IFN-γ production by natural killer cells.

This study defines a critical role for Btk in regulating TLR4-induced crosstalk between antigen presenting cells (APCs) and natural killer (NK) cells. Reduced levels of IL-12, IL-18 and IFN-γ were observed in Btk-deficient mice and ex vivo generated macrophages and dendritic cells (DCs) following acute LPS administration, whilst enhanced IL-10 production was observed. In addition, upregulation of activation markers and antigen presentation molecules on APCs was also impaired in the absence of Btk. APCs, by virtue of their ability to produce IL-12 and IL-18, are strong inducers of NK-derived IFN-γ. Co-culture experiments demonstrate that Btk-deficient DCs were unable to drive wild-type or Btk-deficient NK cells to induce IFN-γ production, whereas these responses could be restored by exogenous administration of IL-12 and IL-18. Thus Btk is a critical regulator of APC-induced NK cell activation by virtue of its ability to regulate IL-12 and IL-18 production in response to acute LPS administration.

Genetics of SLE: functional relevance for monocytes/macrophages in disease.

Genetic studies in the last 5 years have greatly facilitated our understanding of how the dysregulation of diverse components of the innate immune system contributes to pathophysiology of SLE. A role for macrophages in the pathogenesis of SLE was first proposed as early as the 1980s following the discovery that SLE macrophages were defective in their ability to clear apoptotic cell debris, thus prolonging exposure of potential autoantigens to the adaptive immune response. More recently, there is an emerging appreciation of the contribution both monocytes and macrophages play in orchestrating immune responses with perturbations in their activation or regulation leading to immune dysregulation. This paper will focus on understanding the relevance of genes identified as being associated with innate immune function of monocytes and macrophages and development of SLE, particularly with respect to their role in (1) immune complex (IC) recognition and clearance, (2) nucleic acid recognition via toll-like receptors (TLRs) and downstream signalling, and (3) interferon signalling. Particular attention will be paid to the functional consequences these genetic associations have for disease susceptibility or pathogenesis.

Applying random forests to identify biomarker panels in serum 2D-DIGE data for the detection and staging of prostate cancer.

In recent years, Prostate Specific Antigen (PSA) testing is widespread and has been associated with deceased mortality rates; however, this testing has raised concerns of overdiagnosis and overtreatment. It is clear that additional biomarkers are required. To identify these biomarkers, we have undertaken proteomics and metabolomics expression profiles of serum samples from BPH, Gleason score 5 and 7 using two-dimensional difference in gel electrophoresis (2D-DIGE) and nuclear magnetic resonance spectroscopy (NMR). Panels of serum protein biomarkers were identified by applying Random Forests to the 2D-DIGE data. The evaluation of selected biomarker panels has shown that they can provide higher prediction accuracy than the current diagnostic standard. With careful validation of these serum biomarker panels, these panels may potentially help to reduce unnecessary invasive diagnostic procedures and more accurately direct the urologist to curative surgery.

Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms.

Despite the reduced incidence of gastric cancer in the developed world, a diagnosis of stomach carcinoma still carries a poor prognosis due to the asymptomatic nature of the disease in the early stages, subsequent advanced stage diagnosis, and a low 5 year survival rate. Endoscopy remains the primary standard for diagnosis of stomach carcinoma and the current marker, carbohydrate antigen 19-9 (CA19-9) lacks the levels of sensitivity and specificity required in order to make it clinically useful for diagnostic monitoring. Therefore, there is a current need for additional markers to improve the diagnostic accuracy for the early stages of stomach cancer. Together, glycomic, proteomic, and glycoproteomic analyses of serum have the potential to identify such probable markers. A discovery study is reported here using preoperative serum from 80 stomach cancer patients, 10 patients bearing benign stomach disease, and 20 matched controls. Glycomic analysis of the total and immunoaffinity depleted serum revealed statistically significant increases in the levels of sialyl Lewis X epitopes (SLe(X)) present on triantennary glycans accompanied by increased levels of core fucosylated agalactosyl biantennary glycans present on IgG (referred to as the IgG G0 glycoform) which are associated with increasing disease pathogenesis. Protein expression analysis using 2D-DiGE returned a number of differentially expressed protein candidates in the depleted serum, many of which were shown to carry triantennary SLe(X) during subsequent glycomic investigations. Biological pathway analysis of the experimental data returned complement activation and acute phase response signaling as the most significantly altered pathways in the stomach cancer patient serum. Upon the basis of these findings, it is suggested that increased expression of IgG G0 and complement activation are a host response to the presence of the stomach tumor while the increased expression of SLe(X) and acute phase response proteins is a result of pro-inflammatory cytokine signaling, including IL-6, during carcinogenesis. The approach presented herein provides an insight into the underlying mechanisms of disease and the resulting changes in the glycome and glycoproteome offer promise as potential markers for diagnosis and prognostic monitoring in stomach cancer.

Identification of N-glycosylation changes in the CSF and serum in patients with schizophrenia.

Schizophrenia is a major neuropsychiatric disorder that affects 2% of the population worldwide. No biochemical diagnostic tests are available, and patients must undergo lengthy clinical evaluation periods before an accurate diagnosis can be given. Blood and cerebrospinal fluid are candidates for the identification of potential biomarkers for this disease. We have identified several N-glycans that distinguish first onset, unmedicated schizophrenia patients from healthy individuals. This is the first report of the N-glycome from low abundance serum proteins and cerebrospinal fluid. The tetraantennary tetrasialylated glycan with a polylactosamine extension, A4G4LacS4, from low abundance serum proteins showed a 2-fold increase in serum from male schizophrenia patients. Gender specificity was also demonstrated as the triantennary trisialylated glycan containing the SLex epitope was increased significantly in male schizophrenia patients on both high and low abundance serum proteins. Levels of bisecting and sialylated glycans in the cerebrospinal fluid showed a general down-regulation in schizophrenia patients and a 95% positive predictive power for distinguishing patients from controls. These changes are consistent with the reported down-regulation of beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase III and beta-galactoside alpha-2,3/6-sialyltransferases in the prefrontal cortex from schizophrenia patients. These alterations in the N-glycosylation signature could be used potentially for early diagnosis and monitoring of patients after treatment.

Galactosemia, a single gene disorder with epigenetic consequences.

Long-term outcomes of classic galactosemia (GAL) remain disappointing. It is unclear if the complications result mainly from prenatal-neonatal toxicity or persistent glycoprotein and glycolipid synthesis abnormalities. We performed gene expression profiling (T transcriptome) to characterize key-altered genes and gene clusters of four patients with GAL with variable outcomes maintained on a galactose-restricted diet, compared with controls. Significant perturbations of multiple cell signaling pathways were observed including mitogen-activated protein kinase (MAPK) signaling, regulation of the actin cytoskeleton, focal adhesion, and ubiquitin mediated proteolysis. A number of genes significantly altered were further investigated in the GAL cohort including SPARC (osteonectin) and S100A8 (S100 calcium-binding protein). The whole serum N-glycan profile and IgG glycosylation status of 10 treated patients with GAL were compared with healthy control serum and IgG using a quantitative high-throughput analytical HPLC platform. Increased levels of agalactosylated and monogalactosylated structures and decreases in certain digalactosylated structures were identified in the patients. The persistent abnormal glycosylation of serum glycoproteins seen with the microarray data indicates persisting metabolic dyshomeostasis and gene dysregulation in "treated" GAL. Strict restriction of dietary galactose is clearly life saving in the neonatal period; long-term severe galactose restriction may contribute to ongoing systemic abnormalities.

Btk regulates macrophage polarization in response to lipopolysaccharide.

Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\-)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/-) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/-) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/-) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/-) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

Estrogen receptor α regulates tripartite motif-containing protein 21 expression, contributing to dysregulated cytokine production in systemic lupus erythematosus.

OBJECTIVE: To examine the role of 17ß-estradiol in the regulation of the autoantigen tripartite motif-containing protein 21 (TRIM-21) in patients with systemic lupus erythematosus (SLE). METHODS: Monocytes isolated from healthy control subjects and patients with SLE were stimulated with 17ß-estradiol and/or the estrogen receptor α (ERα) antagonist methyl-piperidino-pyrazole dihydrochloride. TRIM-21, ERα, and CREMα expression was determined by real-time polymerase chain reaction (PCR) analysis. MatInspector software was used to identify putative binding sites within the TRIM-21 promoter. ERα binding to the TRIM-21 gene promoter region in monocytes was analyzed by chromatin immunoprecipitation (ChIP) assay. TRIM-21 and interferon regulatory factor 3 protein levels were analyzed by Western blotting. RESULTS: Real-time PCR analysis demonstrated a role of estrogen in the regulation of TRIM-21 expression in monocytes, which correlated positively with ERα gene expression in patients with SLE. Investigations into the human TRIM-21 promoter revealed the presence of an estrogen response element, with ChIP assays confirming ERα binding to this site. Studies into estrogen-induced TRIM-21 expression revealed a hyperresponsiveness of SLE patients to 17ß-estradiol, which led to the enhanced levels of TRIM-21 observed in these individuals. CONCLUSION: Our results demonstrate a role of estrogen in the regulation of TRIM-21 expression through an ERα-dependent mechanism, a pathway that we observed to be overactive in SLE patients. Treatment of monocytes with an ERα antagonist abrogated estrogen-induced TRIM-21 expression and, as a consequence, decreased the expression of interleukin-23. These findings identify TRIM-21 as a novel ERα-regulated gene and provide novel insights into the link between estrogen and the molecular pathogenesis of SLE.

Proteomic analysis of coronary sinus serum reveals leucine-rich α2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure.

BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich α2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P≤0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-α (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-ßR1 (P<0.001) and α-smooth muscle actin (P=0.025) expression. CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, ß-blocker therapy, and BNP.

2D-DIGE as a strategy to identify serum markers for the progression of prostate cancer.

Prostate cancer is the most common solid organ malignancy affecting men in the United States and Western Europe. Currently, the main diagnostic tools used to look for evidence of prostate cancer include physical examination using digital rectal exam (DRE), serum concentrations of prostate specific antigen (PSA) and biopsy. However, due to the low specificity of PSA in differentiating prostate cancer from other benign conditions, many patients undergo overtreatment for their disease. There is an urgent need for additional markers to improve the diagnostic accuracy for early stages of prostate cancer. Proteomic analysis of serum has the potential to identify such markers. An initial discovery study has been completed using 12 serum samples from patients with different grades of prostate cancer (Gleason score 5 and 7) undergoing radical prostatectomy. Serum samples were subjected to immunoaffinity depletion and protein expression analysis using 2D-DIGE. Image analysis isolated 63 spots that displayed differential expression between the Gleason score 5 and 7 cohorts (p < 0.05), 13 of which were identified as statistically significant using two independent image analysis packages. Identification of differentially expressed spots was carried out using LC-MS/MS. Because of their functional relevance and potential significance with regards to prostate cancer progression, two of these proteins, pigment epithelium-derived factor (PEDF) and zinc-alpha2-glycoprotein (ZAG), have undergone extensive validation in serum and tissue samples from the original cohort and also from a larger independent cohort of patients. These results have indicated that PEDF is a more accurate predictor of early stage prostate cancer. We are confident that proteomics-based approaches have the potential to provide more insight into the underlying molecular mechanisms of the disease and also hold great promise for biomarker discovery in prostate cancer.

Fasting status as a consideration for human serum collection and preparation prior to depletion and analysis.

The use of comparative serum proteomic analysis has the potential to reveal protein expression changes present at different stages of disease progression. Depletion strategies allow for the enrichment of low-abundance proteins, which are more likely to be clinically significant biomarkers. We have observed that patient serum samples filtered through 0.22 microm cellulose acetate spin filters prior to depletion showed a variable level of retention of patient material on the upper part of the filter. This could potentially be related to the fasting status of the patient as a reduction in the lipid content of samples through the incorporation of a centrifugation step prior to filtration was found to reduce this effect. In order to determine if proteins were being selectively retained during filtration, a 2-D difference gel electrophoresis (2-D DIGE) experiment was performed. This demonstrated no significant selective retention of protein within crude serum samples. However, as this analysis was carried out on crude serum, it must be emphasised that protein loss could be manifest in the low-abundance proteins which would be masked in our analysis. Depletion of the retentate was not possible due to technical limitations, however based on our results a centrifugation step might act as an alternative to filtration in serum processing prior to depletion.

Urinary markers for prostate cancer.

Prostate cancer is the commonest solid-organ malignancy to affect men in Europe and the USA; it is estimated that one in six men will develop this cancer in their lifetime. Current screening relies on a digital rectal examination with a serum prostate-specific antigen test. Novel urinary diagnostic tests are potentially interesting screening tools for this disease. We examined published reports assessing the use of urinary markers for the diagnosis of prostate cancer. Using a PubMed-based search we identified studies of urinary markers for prostate cancer published from 1985 to February 2006 using the search terms 'urine', 'marker' and 'prostate cancer'. Studies to date have used small cohorts and relied on prostatic biopsies to provide histology. The sensitivity and specificity of markers are wide ranging but with only a few studies published on each putative marker it is difficult to assess their potential impact. Using urinary biomarkers for prostate cancer is a relatively novel diagnostic approach; they are appealing as a screening test because they are not invasive. Further work is needed to identify and validate 'signature markers' indicative of prostatic malignancy. The newer proteomic platforms are promising biomarker discovery tools that might uncover the next generation of urinary biomarkers.