Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-∝

Tumour necrosis factor-a (TNF) can activate NF-?B to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-?B pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment

Background We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor alpha (TNF-alpha) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD).

Design Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab.

Results Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-gamma-secreting Th1 cells and IFN-alpha-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab.

Conclusions New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL23 antibody therapy is a highly effective therapy for these lesions.

FOXP3+ regulatory T-cell counts correlate with histological response in Crohn's colitis treated with infliximab

Crohn's disease is a chronic inflammatory bowel disease of unknown aetiology. Mucosal inflammatory dysregulation is likely important, with increased production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα). The chimeric monoclonal antibody, infliximab, inhibits TNFα and promotes intestinal mucosal healing. Despite this, many patients still require surgical intervention. Patients who have undergone colonic resection post-infliximab therapy, show markedly variable morphological response to treatment. FOXP3+ CD4+ regulatory T-cells have been shown to have a protective role in autoimmune/inflammatory diseases and their sequestration to the bowel is found in those treated with infliximab. We examined the immunohistochemical profile of lymphoid aggregates in tissue sections from post-infliximab Crohn's colitis resection specimens, classified as morphological responders or non-responders, defined in relation to the absence/presence of mucosal ulceration and active inflammation, and a control group. Results indicated no significant diffences in CD68-positive cell counts but increased FOXP3-positive (P = 0.02) and CD4-positive (P = 0.05) cell counts in responders versus non-responders. Untreated control scores were similar to non-responders. Although based on small study numbers, our results suggest an association between upregulation of FOXP3+/CD4+ regulatory T-cells and morphological response to infliximab therapy. This represents a possible quantitative methodology for monitoring therapeutic response to infliximab therapy, based on immunohistochemical evaluation of endoscopic biopsy specimens.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Can emerging biomarkers of myocardial remodelling identify asymptomatic hypertensive patients at risk for diastolic dysfunction and diastolic heart failure?

AIMS: Hypertension is one of the main drivers of the heart failure (HF) epidemic. The aims of this study were to profile fibro-inflammatory biomarkers across stages of the hypertensive heart disease (HHD) spectrum and to examine whether particular biochemical profiles in asymptomatic patients identify a higher risk of evolution to HF.

METHODS AND RESULTS: This was a cross-sectional observational study involving a population of 275 stable hypertensive patients divided into two different cohorts: Group 1, asymptomatic hypertension (AH) (n= 94); Group 2, HF with preserved ejection fraction (n= 181). Asymptomatic hypertension patients were further subdivided according to left atrial volume index ≥34 mL/m(2) (n= 30) and <34 mL/m(2) (n= 64). Study assays involved inflammatory markers [interleukin 6 (IL6), interleukin 8 (IL8), monocyte chemoattractant protein 1 (MCP1), and tumour necrosis factor α], collagen 1 and 3 metabolic markers [carboxy-terminal propeptide of collagen 1, amino-terminal propeptide of collagen 1, amino-terminal propeptide of collagen 3 (PIIINP), and carboxy-terminal telopeptide of collagen 1 (CITP)], extra-cellular matrix turnover markers [matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and tissue inhibitor of metalloproteinase 1 (TIMP1)], and the brain natriuretic peptide. Data were adjusted for age, sex, systolic blood pressure, and creatinine. Heart failure with preserved ejection fraction was associated with an increased inflammatory signal (IL6, IL8, and MCP1), an increased fibrotic signal (PIIINP and CITP), and an increased matrix turnover signal (MMP2 and MMP9). Alterations in MMP and TIMP enzymes were found to be significant indicators of greater degrees of asymptomatic left ventricular diastolic dysfunction.

CONCLUSION: These data define varying fibro-inflammatory profiles throughout different stages of HHD. In particular, the observations on MMP9 and TIMP1 raise the possibility of earlier detection of those at risk of evolution to HF which may help focus effective preventative strategies.

Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H2O2-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 M SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

Apoptosis and cell-cycle regulatory proteins in colorectal carcinoma: relationship to tumour stage and patient survival

The quantitative assessment of apoptotic index (AI) and mitotic index (MI) and the immunoreactivity of p53, bcl-2, p21, and mdm2 were examined in tumour and adjacent normal tissue samples from 30 patients with colonic and 22 with rectal adenocarcinoma. Individual features and combined profiles were correlated with clinicopathological parameters and patient survival data to assess their prognostic value. Increased AI was significantly associated with increased bcl-2 expression (p

Potential improvements in the therapeutic ratio of prostate cancer irradiation: dose escalation of pathologically identified tumour nodules using intensity modulated radiotherapy.

The potential of intensity modulated radiotherapy (IMRT) to improve the therapeutic ratio in prostate cancer by dose escalation of intraprostatic tumour nodules (IPTNs) was investigated using a simultaneous integrated boost technique. The prostate and organs-at-risk were outlined on CT images from six prostate cancer patients. Positions of IPTNs were transferred onto the CT images from prostate maps derived from sequential large block sections of whole prostatectomy specimens. Inverse planned IMRT dose distributions were created to irradiate the prostate to 70 Gy and all the IPTNs to 90 Gy. A second plan was produced to escalate only the dominant IPTN (DIPTN) to 90 Gy, mimicking current imaging techniques. These plans were compared with homogeneous prostate irradiation to 70 Gy using dose–volume histograms, tumour control probability (TCP) and normal tissue complication probability (NTCP) for the rectum. The mean dose to IPTNs was increased from 69.8 Gy to 89.1 Gy if all the IPTNs were dose escalated (p=0.0003). This corresponded to a mean increase in TCP of 8.7–31.2% depending on the /ß ratio of prostate cancer (p

Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNFalpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly, improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo.

Microdialysis enables the chemistry of extracellular ?uid in body tissues to be measured. Extracellular proteases such as the cysteine protease, cathepsin S (CatS), are thought to facilitate astrocytoma invasion. Microdialysates obtained from human brain tumoursin vivo were subjected to cathepsin S activity and ELISA assays. Cathepsin S ELISA expression was detected in ?ve out of 10 tumour microdialysates, while activity was detected in ?ve out of 11 tumour microdialysates. Cathepsin S expression was also detected in microdialysate from the normal brain control although no activity was found in the same sample. While some re?nements to the technique are necessary, the authors demonstrate the feasibility and safety of microdialysis in human astrocytomasin vivo. Characterisation of the extracellular environment of brain tumoursin vivo using microdialysis may be a useful tool to identify the protease pro?le of brain tumours.