Discovery of putative salivary biomarkers for Sjögren's syndrome using high resolution mass spectrometry and bioinformatics

The purpose of the current study was to determine if saliva contains biomarkers that can be used as diagnostic tools for Sj?gren's syndrome (SjS). Twenty seven SjS patients and 27 age-matched healthy controls were recruited for these studies. Unstimulated glandular saliva was collected from the Wharton's duct using a suction device. Two μl of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Raw data were analyzed using bioinformatic tools to identify biomarkers. MALDI O-TOF MS analyses of saliva samples were highly reproducible and the mass spectra generated were very rich in peptides and peptide fragments in the 750-7,500 Da range. Data analysis using bioinformatic tools resulted in several classification models being built and several biomarkers identified. One model based on 7 putative biomarkers yielded a sensitivity of 97.5%, specificity of 97.8% and an accuracy of 97.6%. One biomarker was present only in SjS samples and was identified as a proteolytic peptide originating from human basic salivary proline-rich protein 3 precursor. We conclude that salivary biomarkers detected by high-resolution mass spectrometry coupled with powerful bioinformatic tools offer the potential to serve as diagnostic/prognostic tools for SjS.     

Protein kinase C-theta is required for development of experimental cerebral malaria

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.     

The case for strategic international alliances to harness nutritional genomics for public and personal health

Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countries.     

Snapshots of five clinical ethics committees in the UK

Each of the following papers gives an account of a different UK clinical ethics committee. The committees vary in the length of time they have been established, and also in the main focus of their work. The accounts discuss the development of the committees and some of the ethical problems that have been brought to them. The issues raised will be relevant for other National Health Service (NHS) trusts in the UK that wish to set up such a committee.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

Differential effects of somatostatin and angiopeptin on cell proliferation

1. Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst₂/sst₅ receptor-selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO-K1 cells stably transfected with the human or rat recombinant sst₂ or sst₅ receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors.
2. In CHO-K1 cells, expressing either human or rat recombinant sst₂ or sst₅ receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1–1000 nM) had no effect on basal re-growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml⁻¹) stimulated re-growth of these cells.
3. SRIF (0.1–1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated re-growth in CHO-K1 cells expressing human sst₂ (h sst₂) or sst₅ (h sst₅) receptors (pIC₅₀=8.05±0.03 and 8.56±0.12, respectively). In contrast, angiopeptin (0.1–1000 nM) acted as a partial agonist at the h sst₂ receptor (44.6±2.7% inhibition of the bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.69±0.25) but was devoid of any agonist activity at the h sst₅ receptor.
4. In CHO-K1 cells stably expressing rat recombinant sst₂ (r sst₂) or sst₅ (r sst₅) receptors, SRIF (0.1–1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC₅₀=7.98±24 and 8.50±0.12, respectively). Angiopeptin (0.1–1000 nM) caused a concentration-dependent inhibition of bFGF-stimulated re-growth at the r sst₂ receptor (pIC₅₀=8.08±0.24) but acted as a partial agonist at the r sst₅ receptor (maximum response=57.7±3.6% inhibition of bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.60±0.16).
5. Although angiopeptin was inactive as an agonist at the h sst₅ receptor, 100 nM angiopeptin potently antagonized the SRIF-induced inhibition of proliferation in CHO h  sst₅ (estimated pK_B=10.4±0.3). 5-Hydroxytryptamine (0.1 nM–10 μM) also inhibited bFGF-stimulated re-growth (pIC₅₀=8.36±0.11) and angiopeptin had no effect on this response (pK_B<7).
6. SRIF (0.1–1000 nM) caused a concentration-dependent (pIC₅₀=8.04±0.08) inhibition of bFGF-stimulated re-growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF-stimulated re-growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration-effect curve to SRIF with an estimated pK_B value of 7.70±0.12.
7. These findings suggest that the low intrinsic activity of angiopeptin at the h sst₂ receptor, combined with its lack of agonist activity at the h sst₅ receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

     

Penetrating neck trauma in children: an urban hospital's experience.

OBJECTIVE: As the incidence of violent crime increases in our society, the rate of penetrating head and neck trauma in children also rises. The methods of management of pediatric penetrating neck wounds are addressed. METHODS: All clinical records of children younger than 18 years admitted with penetrating neck injuries between 1990 and 1997 were reviewed. The injuries were classified according to type and location of the neck wound. Demographic data, clinical presentation, diagnostic studies, and management techniques were evaluated. RESULTS: Thirty-five children aged 6 to 18 years old were evaluated for 31 missile wounds and 4 stab wounds. There were 30 boys and 5 girls. Fourteen percent of injuries were in zone 1, 60% in zone II, and 26% in zone III. Of the 33% of children with zone II penetrating neck traumas who underwent selective neck explorations, 86% had significant intraoperative findings. The mortality rates for zones I, II, and III were 60%, 29%, and 56%, respectively. The overall mortality rate was 40%. CONCLUSIONS: Penetrating neck trauma in children may lead to potentially life-threatening injuries. Selective management of penetrating head and neck injuries in children can be a safe and effective policy in an experienced trauma center.