Limited utility of novel serological biomarkers in patients newly suspected of having giant cell arteritis.

AIM: Diagnosing and monitoring vascular activity in giant cell arteritis (GCA) is difficult due to the paucity of specific serological biomarkers. We assessed the utility of 8 novel biomarkers in an inception cohort of newly suspected GCA patients. METHOD: Consecutive patients were enrolled between May 2016 and December 2017. Serum was collected within 72 hours of commencing corticosteroids and at 6 months. It was analyzed for levels of intra-cellular adhesion molecule 1, vascular endothelial growth factor (VEGF), pentraxin 3, von Willebrand factor and procalcitonin (5-plex R&D Systems multiplex assay) and interleukin (IL)6, IL12 and interferon-γ (high-sensitivity 3-plex ProcartaPlex multiplex assay). A GCA specific positron emission tomography / computed tomography (PET/CT) scan was performed at enrolment with uptake in each vascular territory graded and summed to derive a total vascular score (TVS). RESULTS: For the 63 patients enrolled, 12 (19%) had a final diagnosis of biopsy-positive GCA and a further 9 had a clinical diagnosis of biopsy-negative GCA. None of the 8 biomarkers was significantly higher in GCA patients compared with those with alternative diagnoses, or demonstrated a positive correlation with the PET/CT TVS. This was in contrast to the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) which were higher in the biopsy-positive GCA cohort (P < .04) and showed weak positive correlations with the TVS (correlation coefficient 0.34, P < .01). Procalcitonin did not distinguish between GCA and infection. Concentrations of CRP, ESR, VEGF and pentraxin 3 decreased between diagnosis and 6 months in GCA patients. CONCLUSION: This study did not identify new serological biomarkers to assist in diagnosing or assessing the vasculitis burden in GCA.

Skeletal glucocorticoid signalling determines leptin resistance and obesity in aging mice.

OBJECTIVE: Aging and chronic glucocorticoid excess share a number of critical features, including the development of central obesity, insulin resistance and osteoporosis. Previous studies have shown that skeletal glucocorticoid signalling increases with aging and that osteoblasts mediate the detrimental skeletal and metabolic effects of chronic glucocorticoid excess. Here, we investigated whether endogenous glucocorticoid action in the skeleton contributes to metabolic dysfunction during normal aging. METHODS: Mice lacking glucocorticoid signalling in osteoblasts and osteocytes (HSD2OB/OCY-tg mice) and their wild-type littermates were studied until 3, 6, 12 and 18 months of age. Body composition, adipose tissue morphology, skeletal gene expression and glucose/insulin tolerance were assessed at each timepoint. Leptin sensitivity was assessed by arcuate nucleus STAT3 phosphorylation and inhibition of feeding following leptin administration. Tissue-specific glucose uptake and adipose tissue oxygen consumption rate were also measured. RESULTS: As they aged, wild-type mice became obese and insulin-resistant. In contrast, HSD2OB/OCY-tg mice remained lean and insulin-sensitive during aging. Obesity in wild-type mice was due to leptin resistance, evidenced by an impaired ability of exogenous leptin to suppress food intake and phosphorylate hypothalamic STAT3, from 6 months of age onwards. In contrast, HSD2OB/OCY-tg mice remained leptin-sensitive throughout the study. Compared to HSD2OB/OCY-tg mice, leptin-resistant wild-type mice displayed attenuated sympathetic outflow, with reduced tyrosine hydroxylase expression in both the hypothalamus and thermogenic adipose tissues. Adipose tissue oxygen consumption rate declined progressively in aging wild-type mice but was maintained in HSD2OB/OCY-tg mice. At 18 months of age, adipose tissue glucose uptake was increased 3.7-fold in HSD2OB/OCY-tg mice, compared to wild-type mice. CONCLUSIONS: Skeletal glucocorticoid signalling is critical for the development of leptin resistance, obesity and insulin resistance during aging. These findings underscore the skeleton's importance in the regulation of body weight and implicate osteoblastic/osteocytic glucocorticoid signalling in the aetiology of aging-related obesity and metabolic disease.

Red blood cells exposed to cancer cells in culture have altered cytokine profiles and immune function.

It is now accepted that red blood cells (RBCs) from healthy individuals regulate T-cell activity through modulating cytokine interactions, and that stored RBCs or RBCs from inflammatory cohorts are dysfunctional. Our study aimed to investigate how changes in RBCs that have been intentionally modified can affect T-cell activity as a mechanistic test of this modification. Exposure to a cancer cell line in culture was used to alter the cytokine profile of intact RBCs and the effect of these modified RBCs (ccRBCs) on T-cells was evaluated using flow cytometry. We used RBCs from healthy volunteers and quantified cytokines in RBC lysates and conditioned media using Luminex technology. During in vitro cancer cell exposure, RBCs sequestered a variety of cytokines including IL-8, bFGF, and VEGF. Although unmodified RBCs (oRBCs) stimulated proliferation of T-cells (Jurkat cells and peripheral blood mononucleated cells), ccRBCs augmented this proliferative response (3.5-fold and 1.9-fold more respectively). Unlike oRBCs, T-cells stimulated with ccRBCs were no longer protected from phytohemagglutinin-P-driven overexpression of GATA-3 and T-bet and these T-cells were induced to secrete a variety of cytokines including IL-17 and MCP-3. This study supports the hypothesis that RBCs are capable of binding and releasing cytokines in blood, and that modification of these cells can then also affect the T-cell response.

The emerging role of red blood cells in cytokine signalling and modulating immune cells.

For many years red blood cells have been described as inert bystanders rather than participants in intercellular signalling, immune function, and inflammatory processes. However, studies are now reporting that red blood cells from healthy individuals regulate immune cell activity and maturation, and red blood cells from disease cohorts are dysfunctional. These cells have now been shown to bind more than 50 cytokines and have been described as a sink for these molecules, and the loss of this activity has been correlated with disease progression. In this review, we summarise what is currently understood about the role of red blood cells in cytokine signalling and in modulating the activity of immune cells. We also discuss the implications of these findings for transfusion medicine and in furthering our understanding of anaemia of chronic inflammation. By bringing these disparate units of work together, we aim to shine a light on an area that requires significantly more investigation.

Red blood cells are dynamic reservoirs of cytokines.

Red blood cells (RBCs) have been shown to affect immune function and can induce inflammatory responses after transfusion. The transfusion of washed RBCs can significantly reduce adverse effects, however, the soluble factors that may mediate these effects have not been identified. Previous studies have identified, but not quantified, a small number of chemokines associated with RBCs. We isolated RBCs from healthy volunteers and quantified of a panel of 48 cytokines, chemokines, and growth factors in the lysate, cytosol, and conditioned media of these cells using Luminex® technology. This analysis revealed that, after correcting for white blood cell and platelet contamination, 46 cytokines were detected in RBC lysates, and the median concentration in RBCs was 12-fold higher than in the plasma. In addition, extensive washing of RBCs, such as that performed in proteomics analyses or prior to some RBC transfusions, significantly attenuated the release of six cytokines following incubation at 37 °C. This supports the hypothesis that, alongside its gas exchange function, RBCs play a role in cytokine signalling. This discovery may help supplement disease biomarker research and may shed light on adverse inflammatory processes that can follow RBC transfusion.

Red blood cells: The primary reservoir of macrophage migration inhibitory factor in whole blood.

Red blood cells are widely accepted to be inert carriers of oxygen and haemoglobin, but there is growing evidence that they play a much more critical role in immune function. Macrophage migration inhibitory factor (MIF) is a key cytokine in disease with additional oxido-reductase activity, which aids in managing oxidative stress. Although two studies have reported the presence of MIF in red blood cells, no study has quantified the levels of this protein. In this study, freshly isolated plasma, platelets, leukocytes, and red blood cells from healthy individuals were collected and the concentration of MIF was determined using an enzyme linked immunosorbent assay. This analysis demonstrated that MIF in red blood cells was present at 25 µg per millilitre of whole blood, which is greater than99% of the total MIF and 1000-fold higher concentration than plasma. This result was supported by electrophoresis and Western blot analysis, which identified MIF in its monomer structural form following sample processing. Furthermore, by assessing the level of tautomerase activity in red blood cell fractions in the presence of a MIF inhibitor, it was determined that the red blood cell-derived MIF was also functionally active. Together, these findings have implications on the effect of haemolysis during sample preparation and provide some clue into the inflammatory processes that occur following haemolysis in vivo. These results support the hypothesis that red blood cells are a major reservoir of this inflammatory protein and may play a role in inflammation.

Effect of Laparoscopic Sleeve Gastrectomy on Fasting Gastrointestinal, Pancreatic, and Adipose-Derived Hormones and on Non-Esterified Fatty Acids.

BACKGROUND: Alterations in gastrointestinal, pancreatic, and adipose hormone levels may have a greater role in weight loss than initially appreciated. The laparoscopic sleeve gastrectomy (LSG) operation is now the most frequently performed bariatric operation in many countries, but there are relatively few data regarding its molecular effects. We sought to characterize the effect of LSG on fasting plasma levels of selected hormones and on non-esterified fatty acids (NEFA), and to compare these to levels in non-obese control individuals. MATERIALS AND METHODS: The levels of nine plasma hormones were measured using a multiplex bead-based assay at baseline and at 3 months after operation in 11 obese patients undergoing LSG. NEFA levels were also measured. The levels were compared to those for 22 age- and sex-matched non-obese individuals. RESULTS: At baseline, obese patients showed significantly higher expression of C-peptide, insulin, and leptin and significantly lower ghrelin, glucose-dependent insulinotropic peptide (GIP), and resistin compared to non-obese controls (p < 0.05). LSG resulted in a reduction in BMI from 42.5 ± 6.47 kg/m2 at operation to 35.2 ± 5.14 kg/m2 at 3 months (42 % mean excess weight loss, p < 0.001). LSG led to a significant decrease in ghrelin, glucagon-like peptide-1 (GLP-1), glucagon, leptin, plasminogen activator inhibitor-1 (PAI-1), and NEFA. CONCLUSION: LSG induces marked early changes in the fasting levels of factors thought to be important regulators of obesity and metabolic health. These changes differ somewhat from the findings for operations with a malabsorptive component, suggesting that subtle differences exist in the mechanisms of weight loss between LSG and other bariatric operations.

Subcutaneous fat transplantation alleviates diet-induced glucose intolerance and inflammation in mice.

AIMS/HYPOTHESIS: Adipose tissue (AT) distribution is a major determinant of mortality and morbidity in obesity. In mice, intra-abdominal transplantation of subcutaneous AT (SAT) protects against glucose intolerance and insulin resistance (IR), but the underlying mechanisms are not well understood. METHODS: We investigated changes in adipokines, tissue-specific glucose uptake, gene expression and systemic inflammation in male C57BL6/J mice implanted intra-abdominally with either inguinal SAT or epididymal visceral AT (VAT) and fed a high-fat diet (HFD) for up to 17 weeks. RESULTS: Glucose tolerance was improved in mice receiving SAT after 6 weeks, and this was not attributable to differences in adiposity, tissue-specific glucose uptake, or plasma leptin or adiponectin concentrations. Instead, SAT transplantation prevented HFD-induced hepatic triacylglycerol accumulation and normalised the expression of hepatic gluconeogenic enzymes. Grafted fat displayed a significant increase in glucose uptake and unexpectedly, an induction of skeletal muscle-specific gene expression. Mice receiving subcutaneous fat also displayed a marked reduction in the plasma concentrations of several proinflammatory cytokines (TNF-α, IL-17, IL-12p70, monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1ß [ΜIP-1ß]), compared with sham-operated mice. Plasma IL-17 and MIP-1ß concentrations were reduced from as early as 4 weeks after transplantation, and differences in plasma TNF-α and IL-17 concentrations predicted glucose tolerance and insulinaemia in the entire cohort of mice (n = 40). In contrast, mice receiving visceral fat transplants were glucose intolerant, with increased hepatic triacylglycerol content and elevated plasma IL-6 concentrations. CONCLUSIONS/INTERPRETATION: Intra-abdominal transplantation of subcutaneous fat reverses HFD-induced glucose intolerance, hepatic triacylglycerol accumulation and systemic inflammation in mice.

A preliminary report on stem cell therapy for neuropathic pain in humans.

OBJECTIVE: Mesenchymal stem cells (MSCs) have been shown in animal models to attenuate chronic neuropathic pain. This preliminary study investigated if: i) injections of autologous MSCs can reduce human neuropathic pain and ii) evaluate the safety of the procedure. METHODS: Ten subjects with symptoms of neuropathic trigeminal pain underwent liposuction. The lipoaspirate was digested with collagenase and washed with saline three times. Following centrifugation, the stromal vascular fraction was resuspended in saline, and then transferred to syringes for local injections into the pain fields. Outcome measures at 6 months assessed reduction in: i) pain intensity measured by standard numerical rating scale from 0-10 and ii) daily dosage requirements of antineuropathic pain medication. RESULTS: Subjects were all female (mean age 55.3 years ± standard deviation [SD] 14.67; range 27-80 years) with pain symptoms lasting from 4 months to 6 years and 5 months. Lipoaspirate collection ranged from 102-214 g with total cell numbers injected from 33 million to 162 million cells. Cell viability was 62%-91%. There were no systemic or local tissue side effects from the stem cell therapy (n=41 oral and facial injection sites). Clinical pain outcomes showed that at 6 months, 5/9 subjects had reduced both pain intensity scores and use of antineuropathic medication. The mean pain score pre-treatment was 7.5 (SD 1.58) and at 6 months had decreased to 4.3 (SD 3.28), P=0.018, Wilcoxon signed-rank test. Antineuropathic pain medication use showed 5/9 subjects reduced their need for medication (gabapentin, P=0.053, Student's t-test). CONCLUSION: This preliminary open-labeled study showed autologous administration of stem cells for neuropathic trigeminal pain significantly reduced pain intensity at 6 months and is a safe and well tolerated intervention.

Diversity of microbial species implicated in keratitis: a review.

BACKGROUND: Microbial keratitis is an infectious disease of the cornea characterised by inflammation and is considered an ophthalmic emergency requiring immediate attention. While a variety of pathogenic microbes associated with microbial keratitis have been identified, a comprehensive review identifying the diversity of species has not been completed. METHODS: A search of peer-reviewed publications including case reports and research articles reporting microorganims implicated in keratitis was conducted. Search engines including PubMed, Scopus and Web of Science with years ranging from 1950-2012 were used. RESULTS: 232 different species from 142 genera, representing 80 families were found to be implicated in microbial keratitis. Fungi exhibited the largest diversity with 144 species from 92 genera. In comparison, 77 species of bacteria from 42 genera, 12 species of protozoa from 4 genera and 4 types of virus were identified as the infectious agents. A comparison of their aetiologies shows reports of similarities between genera. CONCLUSIONS: The diversity of microbial species implicated in keratitis has not previously been reported and is considerably greater than suggested by incidence studies. Effective treatment is heavily reliant upon correct identification of the responsible microorganisms. Species identification, the risk factors associated with, and pathogenesis of microbial keratitis will allow the development of improved therapies. This review provides a resource for clinicians and researchers to assist in identification and readily source treatment information.