Pharmaceutical/food grade titanium dioxide particles are absorbed into the bloodstream of human volunteers.

BACKGROUND: Exposure to persistent engineered nano and micro particles via the oral route is well established. Animal studies have demonstrated that, once ingested, a small proportion of such particles translocate from the gastrointestinal tract to other tissues. Exposure to titanium dioxide is widespread via the oral route, but only one study has provided indirect evidence (total titanium analyses) of absorption into the blood stream in humans. We sought to replicate these observations and to provide additional evidence for particulate uptake. FINDINGS: Human volunteers with normal intestinal permeability were orally administered 100 mg pharmaceutical/food grade titanium dioxide. Blood samples were collected from 0.5 to 10 h post ingestion and analysed for the presence of reflectant bodies (particles) by dark field microscopy, and for total titanium by inductively coupled plasma mass spectrometry (ICP-MS). Blood film analyses implied early absorption of particles (2 h) with a peak maximum at 6 h following ingestion. The presence of these reflectant particles in blood roughly mirrored the levels of total titanium by ICP-MS, providing good evidence for the latter being a measure of whole particle (titanium dioxide) absorption. CONCLUSIONS: This study shows that a fraction of pharmaceutical/food grade titanium dioxide is absorbed systemically by humans following ingestion. It confirms that at least two routes of particle uptake may exist in the human gut- one proximal and one distal. Further work should quantify human exposure and uptake of such persistent particles.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells.

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

Assessment of Integration-defective HIV-1 and EIAV Vectors In Vitro and In Vivo.

The interest in integrase-defective lentiviral vectors (IDLVs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV) to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector.Molecular Therapy - Nucleic Acids (2012) 1, e60; doi:10.1038/mtna.2012.53; published online 11 December 2012.

A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.

ProSavin is an equine infectious anemia virus vector-based gene therapy for Parkinson's disease for which inducible HEK293T-based producer cell lines (PCLs) have been developed. These cell lines demonstrate stringent tetracycline-regulated expression of the packaging components and yield titers comparable to the established transient production system. A prerequisite for the use of PCL-derived lentiviral vectors (LVs) in clinical applications is the thorough characterization of both the LV and respective PCL with regard to identity and genetic stability. We describe the detailed characterization of two ProSavin PCLs (PS5.8 and PS46.2) and resultant ProSavin vector. The two cell lines demonstrate stable production of vector over a time period sufficient to allow generation of master and working cell banks, and subsequent large-scale vector production. ProSavin generated from the PCLs performs comparably in vivo to that produced by the standard transient transfection process with respect to transduction efficiency and immunogenicity. The development of ProSavin PCLs, and the detailed characterization described here, will aid the advancement of ProSavin for clinical application.

Dietary microparticles and their impact on tolerance and immune responsiveness of the gastrointestinal tract.

Dietary microparticles are non-biological bacterial-sized particles of the gastrointestinal lumen that occur due to endogenous formation (calcium phosphate) or following oral exposure (exogenous microparticle). In the UK, about 40 mg (10(12)) of exogenous microparticles are ingested per person per day, through exposure to food additives, pharmaceutical/supplement excipients or toothpaste constituents. Once ingested, exogenous microparticles are unlikely to pass through the gastrointestinal tract without adsorbing to their surfaces some ions and molecules of the intestinal lumen. Both entropy and ionic attraction drive such interactions. Calcium ions are especially well adsorbed by dietary microparticles which then provide a positively charged surface for the attraction (adsorption) of other organic molecules such as lipopolysaccharides, peptidoglycans or protein antigen from the diet or commensal flora. The major (but not only) sites of microparticle entry into intestinal tissue are the M-cell rich lymphoid aggregates (termed Peyer's patches in the small bowel). Indeed, it is well established that this is an efficient transport route for non-biological microparticles although it is unclear why. We hypothesise that this pathway exists for "endogenous microparticles" of calcium phosphate, with immunological and physiological benefit, and that "exogenous dietary microparticles", such as titanium dioxide and the silicates, hijack this route. This overview focuses on what is known of these microparticles and outlines their potential role in immune tolerance of the gut (endogenous microparticles) or immune activation (exogenous microparticles) and inflammation of the gut.

Low dietary calcium levels modulate mucosal caspase expression and increase disease activity in mice with dextran sulfate sodium induced colitis.

Dietary calcium (Ca) positively modulates the susceptibility to colon cancer, but its effects on related or earlier colonic pathologies, such as inflammation and mucosal dysregulation, are poorly understood. We tested the effects of differing dietary Ca levels on acute dextran sulfate sodium (DSS)-induced colitis in mice. BALB/c mice received a normal Ca (NCa) diet (0.5% Ca), a high Ca (HCa) diet (1.5% Ca), a low Ca (LCa) diet (0.05% Ca), or a very low Ca (VLCa) diet (0.009% Ca) for 3 wk. Mucosal caspases 1, 3, and 9 were assessed by Western blotting, and the histological crypt score was assessed by microscopy. Half of the mice in each group received DSS (1.5%) for 20 d in their drinking water, and disease activity was assessed. Increasing or lowering dietary Ca increased mucosal caspases (P < 0.0001 vs. NCa). Crypt scores increased with decreasing dietary Ca levels (P < 0.0001, r = -0.675), indicating that elevated caspases in LCa groups reflected early subclinical inflammation. DSS-induced disease activity was higher in mice fed low dietary Ca levels [P < 0.0001, VLCa and DSS vs. NCa and DSS (NCaDSS) and P < 0.005, LCa and DSS vs. NCaDSS], and mice from the VLCa group were moribund within 11 d of DSS administration. Those in the HCa group did not differ greatly from controls. Together, these data indicate that Ca protects against DSS-induced colitis in mice. The mechanisms are unclear, but the calcium-sensing receptor and/or luminal precipitates of calcium phosphate microparticles may be involved. Whether these observations can be extended to patients with colitis or infectious diarrhea deserves consideration.