SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies.

The past decade has seen the emergence of multidrug resistant pathogens as a leading cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including host range alterations, constitutive lytic growth, and control over phage replication. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to this problem, we propose the SpyPhage system where a "scaffold" bacteriophage is engineered to incorporate a SpyTag moiety on its capsid head to enable rapid postsynthetic modification of their surfaces with SpyCatcher-fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and whole genome assembly, we targeted a SpyTag capsid fusion to K1F, a phage targeting the pathogenic strain Escherichia coli K1. We demonstrate for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying "scaffold" genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations.

The significance of coeliac disease antibodies in patients with ankylosing spondylitis: a case-controlled study.

The presence of anti-gliadin antibodies (AGA) and their relationship with intestinal permeability and prevalence of undiagnosed coeliac disease (CD) in ankylosing spondylitis (AS) were investigated. Blood samples from 30 AS patients and 19 age- and sex-matched controls were analysed for human leucocyte antigen (HLA)-B27, AGA and endomysial antibodies (EMA). Immunoglobulin (Ig) A-type AGA and IgG-type EMA were determined by enzyme-linked immunosorbent assay. AGA-positive patients were examined by gastroduodenoscope and proximal small bowel mucosa biopsies were performed. Eleven (36.7%) AS patients were AGA positive (compared with none of the control subjects) and three (10.0%) of these AS patients were also EMA-positive. The presence of AGA was not associated with more severe AS. Mild-to-severe villous atrophy and hyperplasia of crypts with increased chronic inflammatory cells in the lamina propria, which is typical of CD, was only observed in one AGA/EMA positive AS patient; CD was subsequently diagnosed by histology. Although AGA positivity might contribute to the pathogenesis of AS by increasing intestinal permeability to micro-organisms or by modifying intestinal immune mechanisms, further work is required to clarify its role in AS.

Plasma osteoprotegerin concentrations in type 2 diabetic patients and its association with neuropathy.

BACKGROUND AND AIMS: To investigate the association of plasma osteoprotegerin (OPG) levels with diabetic neuropathy. METHODS: Forty-two diabetic patients (21 female and 21 male) and twenty-four non-diabetic healthy control subjects (12 female and 12 male) were included in the study. All consecutive diabetic patients who came for routine follow-up at our outpatient clinic were invited to participate in this clinical study. We studied EMG and neuropathy symptom score in all study subjects. Fasting plasma glucose, HbA1 C, hs-CRP, OPG levels and lipid profile were measured for each subject. RESULTS: Serum fasting glucose, HbA1c, HOMA-IR, total cholesterol, triglyserid, LDL-Cholesterol, HDL-Cholesterol, lipoprotein (a), apolipoprotein-b, hs-CRP, OPG levels, and neuropathy symptom score were statistically higher in diabetic patients than in healthy control subjects. Plasma OPG levels was statistically higher in diabetic patients than it was in nondiabetic control subjects. However, plasma OPG levels were not significantly different between diabetic patients without neuropathy and healthy control subjects. On the other hand, OPG levels were statistically higher in diabetic patients with neuropathy than in patients without neuropathy. In addition to that serum fasting glucose, HbA1c, hs-CRP, diabetes duration, neuropathy symptom score were statistically higher in diabetic patients with neuropathy than they were in patients without neuropathy. In total group of subjects, plasma OPG levels were correlated significantly with age, diabetes duration, HbA1c, total cholesterol, HDL-cholesterol, lipoprotein (a), apolipoprotein b, hs-CRP. In diabetic patients, plasma OPG correlated significantly with age, diabetes duration, neuropathy symptom score, HbA1c, lipoprotein (a), apolipoprotein b levels. CONCLUSIONS: The major findings of this study were that the plasma OPG concentrations were higher in type 2 diabetic patients than OPG concentrations in healthy control subjects and they were positively correlated with diabetic neuropathy. This finding supports the growing concept that OPG acts as an important regulator in the development of vascular dysfunction in diabetes.

The relationship between left ventricular mass index and insulin sensitivity, postprandial glycaemia, and fasting serum triglyceride and adiponection levels in patients with type 2 diabetes.

The relationship between left ventricular mass index (LVMI) and insulin sensitivity, postprandial glycaemia, fasting serum triglyceride and adiponectin was investigated in 70 patients with type 2 diabetes. Serum fasting insulin, C-peptide, high-sensitivity C-reactive protein (hs-CRP), glycated haemoglobin (HbA1c), postprandial glycaemia, lipids and fasting serum adiponectin levels were measured. Ventricular hypertrophy was assessed at rest by electrocardiography and echocardiography. Insulin sensitivity was assessed using the homeostasis model assessment index (HOMA-IR). LVMI was assessed using the Devereux formula. Study patients had lower than normal HOMA-IR, and higher than normal serum fasting insulin levels and LVMI, and tended to have reduced insulin sensitivity. Pearson's correlation coefficient showed a statistically significant correlation between fasting serum adiponectin and LVMI, fasting serum insulin, HOMA-IR, HbA1c, serum postprandial glucose and hs-CRP. There were no statistically significant correlations between LVMI and serum hs-CRP or HOMA-IR. The results indicate the importance of fasting serum adiponectin in the development of cardiovascular complications, such as increased LVMI.

Does rofecoxib increase TNF-alpha levels?

OBJECTIVE: Rofecoxib (Vioxx), the first COX-2 selective non-steroidal anti-inflammatory drug (NSAID), was recently withdrawn from the market due to the increased risk of acute myocardial infarction. The precise mechanism responsible for this phenomenon still remains unknown. Tumor necrosis factor alpha (TNF-alpha) is a cytokine, possibly most responsible for mortality in patients with acute myocardial infarction. However, this study was designed to study possible effects of rofecoxib on the level of TNF-alpha by using MSU crystal induced inflammation in the rat subcutaneous air pouch model. METHODS: Rat subcutaneous air pouches were produced and examinations commenced 6 days later. Control groups received only MSU crystals, or no crystals or drugs. To begin with, rofecoxib (30 mg/kg), indomethacin (20 mg/kg) or diclofenac (3 mg/kg) were administered to groups of 5 rats each. Thirty minutes later, MSU crystals were injected into air pouches, except for the negative control group. Twenty-four hours later, the rats were sacrificed for aspiration of fluid and for the dissection of pouch walls to determine leukocyte counts, pouch wall histology, and to assay IL-10 and TNF-alpha. RESULTS: Intra-pouch injection of MSU crystals, compared to non-injected pouches, caused an increase in white blood cell count (WBC) (30 +/- 44.7 versus 4508 +/- 792.3 cells/mm3), in the numbers of pouch wall vessels (vascular index) (4.8 +/- 0.3 versus 11.4 +/- 1.5 vessels/high-power field) and in TNF-alpha (50.0 +/- 13.4 versus 70.34 +/- 20.9 ng/mL), but not in interleukin-10 (IL-10) (60.6 +/- 63.0versus 61.48 +/- 7.1). WBC and vascular index were significantly reduced in all study groups compared to the control group (p < 0.05). Levels of TNF- in fluids were unexpectedly and significantly (p < 0.05) increased in all cases. The highest level of TNF-alpha was found in the rofecoxib group. In contrast to TNF-alpha, IL-10 levels were significantly (p < 0.05) decreased in all three drug groups. Indomethacin tended to suppress inflammation more effectively. However, there was no significant difference between the groups for IL-10 (p > 0.05). CONCLUSION: All three NSAIDs exhibited anti-inflammatory activity against MSU crystal induced inflammation. The difference in anti-inflammatory effects of these three non-steroidal drugs is seen not only in the anti-inflammatory effect on MSU induced inflammation but also in the nature of the effects. Refocoxib tended to increase the TNF-alpha level. Whether increased TNF-alpha levels can help explain the side effect of COX-2 specific inhibitors still requires further studies.