Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages.

BACKGROUND AND PURPOSE: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo. EXPERIMENTAL APPROACH: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation. KEY RESULTS: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2. CONCLUSIONS AND IMPLICATIONS: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.

The effect on visual function of Hydroview intraocular lens opacification: a cross-sectional study.

PURPOSE: Hydroview intraocular lenses (IOLs) have been associated with symptomatic opacification of the optic necessitating IOL exchange. Glare and misty vision have been noted as common presenting symptoms. This study's purpose was to investigate the impact of IOL opacification on objective measurements of visual function, including glare, and on vision-related quality of life. METHODS: All patients who underwent Hydroview IOL implantation at Bristol Eye Hospital between December 2000 and the end of February 2001 were invited for assessment along with patients found to have Hydroview IOL opacification in routine ophthalmic clinics. Glare, visual acuity, contrast sensitivity, visual field, and colour vision were assessed. Vision-related quality of life and subject's symptoms were determined by questionnaire. IOL opacification was assessed by slit lamp bio-microscopy and anterior segment photography. RESULTS: Data from 129 patients were analysed. Fifty subjects had opacified IOLs and 79 clear IOLs. Subjects with opacified IOLs showed dramatically higher levels of glare (adjusted mean difference of 0.63 log units 95% CI, 0.45-0.82; P<0.001) with only mildly reduced visual acuity (adjusted mean difference of 0.09 logMAR units 95% CI, 0.03-0.15; P=0.002). Opacification was associated with poorer contrast sensitivity (P<0.001), visual field (P<0.001), and with lower vision-related quality of life (P<0.001). CONCLUSIONS: This study highlights the significant impact IOL opacification has on visual performance and experience, in particular glare and consequent impact on quality of life. The study shows that to quantify accurately the effect of IOL opacification on vision glare must be assessed.

The Cataract National Dataset electronic multicentre audit of 55,567 operations: when should IOLMaster biometric measurements be rechecked?

PURPOSE: Calculation of intraocular lens (IOL) power for implantation during cataract surgery depends on ocular biometric measurements. The aim of this study was to characterise the normal range of intra- and interindividual variation in axial length (AL) and corneal power (K) when IOLMaster measurements were possible and to derive recommendations as to which outlying measurements merit verification before acceptance. METHODS: The Medisoft electronic patient database contains prospectively collected data conforming to the United Kingdom (UK) Cataract National Dataset on 55,567 cataract operations. From this AL and K information on the 32,556 eyes (14,016 paired) of patients older than 25 years, without corneal pathology, history of intraocular surgery and who had all biometric measurements taken with the Zeiss IOLMaster (Carl Zeiss Meditec) were extracted. R 2.8.1 (R Foundation for Statistical Computing) was used for statistical analysis. RESULTS: Mean age was 76.4 years and 62.0% were female. Mean (95% confidence interval) values for AL, mean K and corneal astigmatism were 23.40 (21.27-26.59) mm, 43.90 (40.94-47.01) D and 1.04 (<2.50) D. Nearly all astigmatism was either with or against the rule. Differences between paired eyes were not statistically significant. 95% individuals had asymmetry of AL and mean K<0.70 mm and 0.92 D, respectively. CONCLUSIONS: On the basis of approximation of the 95% CI above, it is suggested that AL, mean K and keratometric astigmatism measurements outside the ranges 21.30-26.60 mm, 41.00-47.00 D and >2.50 D, respectively, and intraindividual asymmetry of AL >0.70 mm or mean K>0.90 D should be verified before acceptance.

Role of pattern-recognition receptors in cardiovascular health and disease.

A role for PRRs (pattern-recognition receptors) in immune cell function is now well established. In macrophages and other immune cells, activation of TLRs (Toll-like receptors) and cytosolic NLRs [NOD (nucleotide oligomerization domain) proteins containing a leucine-rich repeat] results in the induction of genes and release of imunoregulator hormones including cytokines and NO (nitric oxide). In addition to immune cells, structural cells of the cardiovascular system including endothelial cells, vascular smooth muscle and cardiac myocytes express functional PRRs and sense PAMPs (pathogen-associated molecular patterns). Furthermore, bacteria and PAMPs activate the coagulation system and platelets. TLRs are now implicated in a range of cardiovascular diseases and syndromes including atherosclerosis and sepsis. Our group is working on the hypotheses that differences exist in how tissues of the cardiovascular system, including vessels, endothelium, heart and blood, sense pathogens compared with immune cells (principally macrophages) and that identifying such differences will reveal new therapeutic targets for the treatment of cardiovascular disease. We have identified examples of similarities and differences in how cardiovascular tissues and macrophages sense PAMPs. These findings will be discussed together with our interpretation of how this information may lead to new treatments.

Effect and duration of lung volume reduction surgery: mid-term results of the Brompton trial.

UNLABELLED: Although many studies have reported improvement in lung function following LVRS, the magnitude of improvement and subsequent decline has not been evaluated against medical therapy after the second year. METHODS: Existing pulmonary function records were collapsed for ech participant since randomisation from Brompton LVRS trial cohort. Longitudinal data analysis was used to profile th history of medically treated patients and the effect of LVRS. RESULTS: Pulmonary function results were collated from survivors over a median of 25 (17 to 39) months. The estimated immediate increase in mean FEV1, following surgery was +0.2591 (0.179, 0.339), with a rate of change of -0.0051 (-0.009, -0.001) per month compared to medical therapy (p < 0.001). The changes in the secondary outcome measures (LVRS compared to medical therapy) were an increase in FVC (p = 0.004), decrease in RV (p < 0.001) and TLC (p < 0.001), with differences that were maintained over time. The initial reduction in RV/TLC ration was sustained (p < 0.001), but the estimated initial increase in peak flow was accompanied by a gradual decline that was not statistically significant (p = 0.062). KCOc showed no immediate change, but there was a gradual sustained increase with time (p = 0.009). Mean oxygen saturations improved and continued to do so compared to patients on medical therapy (p = 0.001). CONCLUSIONS: The immediate increase in FEV1 is not sustained, although the mechanical improvements of LVRS on increasing FVC, reducing both the RV and RV/TLC ratio, appear to be maintained. The important benefits of LVRS may be the gradual and sustained increase in transfer factor accompanied by improved oxygen saturations.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation.

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

Cytokine secretion in mixed lymphocyte culture: a prognostic indicator of renal allograft rejection in addition to HLA mismatching.

We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.

A study of cytokine protein secretion, frequencies of cytokine expressing cells and IFN-G gene polymorphisms in normal individuals.

BACKGROUND: Cytokines are major regulators of immune responses, and there is evidence that they play a role in allograft rejection. Before embarking on a detailed study of pretransplant cytokine profiles in renal allograft recipients, we wished to investigate variations in cytokine protein secretion, numbers of cytokine expressing T cells, and cytokine gene polymorphisms in normal volunteers. METHODS: Twenty normal healthy volunteers were studied. Cytokine protein secretion [interleukin- (IL) 2, IL-4, IL-10, and interferon- (IFN) y] and numbers of cytokine expressing CD3+ T cells (IL-2, IL-4, IL-10, and IFN-gamma) were quantified by means of enzyme-linked immunosorbent assay and two-color flow cytometry respectively. IFN-gamma gene polymorphisms were determined by polymerase chain reaction and autoradio graphy. RESULTS: Large interindividual variations in both the quantity of IL-2, IL-4, IL-10, and IFN-gamma cytokine protein secreted and numbers of IL-2 and IFN-gamma expressing T cells were demonstrated. However, numbers of IL-4 and IL-10 expressing cells were found to be below detectable limits by flow cytometry. In the case of IFN-gamma, a bi-modal distribution was seen for the quantity of protein secreted. In addition, correlations were observed between IL-2 protein and frequency of IL-2 expressing T cells. However, no relationship was found between IFN-gamma protein levels, numbers of IFN-gamma expressing cells and IFN-gamma gene polymorphisms. CONCLUSIONS: We have demonstrated large differences in both numbers of T helper 1 cytokine expressing cells and the quantity of T helper 1 and T helper 2 cytokine protein secreted between normal individuals. Although the amount of IL-2 protein secreted appeared to be determined by the frequency of IL-2 expressing cells, this was not the case for IFN-gamma.

Detection of human papillomavirus in vulval carcinoma using semi-nested PCR and restriction enzyme typing: a rapid and sensitive technique.

Aims-To develop a highly sensitive technique for the reliable detection and typing of human papillomavirus (HPV) DNA in clinical tissue.Methods-A two step, semi-nested PCR was used with primers spanning the L1 region of the HPV genome and capable of detecting HPV DNA of all known HPV types. The clinical samples were typed by digestion of the 412 base pair PCR product with Rsa I, generating unique fragments for each HPV type. Thirteen samples were screened by this method, including nine vulval carcinoma samples and four wart samples from the penis and vulva.Results-Experiments using DNA extracted from HPV DNA positive cell lines-that is, CaSki (HPV type 16) and HeLa (HPV type 18) established that the technique could detect as few as 50 HPV copies and that the predicted Rsa I fragments from HPV types 16 and 18 were generated. The predicted 412 base pair fragment was observed for all 13 clinical samples subjected to semi-nested PCR. Rsa I digestion of the product of the second round of PCR permitted the positive identification of the HPV type in most cases.Conclusions-This technique provides an effective and rapid means of detecting HPV DNA, in most cases providing the HPV type. High risk HPV types were always detected in the nine vulval carcinoma samples analysed. The amount of tissue available from the biopsy specimens was small, confirming the sensitivity of the method.

Bacterial attack on phenolic ethers. Purification and characterization of the components of the meta O-dealkylase of Pseudomonas fluorescens Tp.

The meta O-dealkylase of Pseudomonas fluorescens Tp has been resolved into two protein components, neither of which is a cytochrome. The substrate binding terminal oxidase has been purified and shown to be a non-haem iron protein of approximate molecular weight 118,000, consisting of two seemingly identical subunits, each of molecular weight 55,000. Binding of substrate by the terminal oxidase has been established by difference spectroscopy. The amino acid composition of the protein has also been determined. The NADH-dependent reductase of the system has been partly purified and appears to have a molecular weight of 80,000. The similarity between this and other bacterial O-dealkylases is discussed.