Impaired neutrophil directional chemotactic accuracy in chronic periodontitis patients

Aim: To investigate the chemotactic accuracy of peripheral blood neutrophils from patients with chronic periodontitis compared with matched healthy controls, before and after non-surgical periodontal therapy. Material & Methods: Neutrophils were isolated from patients and controls (n = 18) by density centrifugation. Using the Insall chamber and video microscopy, neutrophils were analysed for directional chemotaxis towards N-formyl-methionyl-leucyl-phenylalanine [fMLP (10 nM), or CXCL8 (200 ng/ml)]. Circular statistics were utilized for the analysis of cell movement. Results: Prior to treatment, neutrophils from patients with chronic periodontitis had significantly reduced speed, velocity and chemotactic accuracy compared to healthy controls for both chemoattractants. Following periodontal treatment, patient neutrophils continued to display reduced speed in response to both chemoattractants. However, velocity and accuracy were normalized for the weak chemoattractant CXCL8 while they remained significantly reduced for fMLP. Conclusions: Chronic periodontitis is associated with reduced neutrophil chemotaxis, and this is only partially restored by successful treatment. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to their disease by increasing tissue transit times, thus exacerbating neutrophil-mediated collateral host tissue damage

Potentiation of Epithelial Innate Host Responses by Intercellular Communication

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection

Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori

Bacterial-mediated cancer therapy has shown great promise in in vivo tumour models with increased survival rates post-bacterial treatment. Improving efficiency of bacterial-mediated tumour regression has focused on controlling and exacerbating bacterial cytotoxicity towards tumours. One mechanism that has been used to carry this out is the process of bactofection where post-invasion, bacteria deliver plasmid-borne mammalian genes into target cells for expression. Here we utilised the cancer-targeting Salmonella Typhimurium strain, SL7207, to carry out bactofection into triple negative breast cancer MDA-MB-231 cells. However, we noted that post-transformation with the commonly used mammalian expression vector pEGFP, S. Typhimurium became filamentous, attenuated and unable to invade target cells efficiently. Filamentation did not occur in Escherichia coli-transformed with the same plasmid. Further investigation identified the region inducing S. Typhimurium filamentation as being the f1 origin of replication (f1 ori), an artefact of historic use of mammalian plasmids for single stranded DNA production. Other f1 ori-containing plasmids also induced the attenuated phenotype, while removal of the f1 ori from pEGFP restored S. Typhimurium virulence and increased the bactofection capacity. This work has implications for interpretation of prior bactofection studies employing f1 ori-containing plasmids in S. Typhimurium, while also indicating that future use of S. Typhimurium in targeting tumours should avoid the use of these plasmids

Phagocytic Activity and Oxidative Burst of Granulocytes in Persons with Myeloperoxidase Deficiency

Peer Reviewe