The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo.

BACKGROUND: The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO 2 NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO 2 NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO 2 NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO 2 NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo. METHODS: Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO 2 NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO 2 NP, OVA plus rutile TiO 2 NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated. RESULTS: All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile

The nearest neighbor nuclei method to objectify analysis of pertussis toxin-induced clustering.

The in vivo histamine sensitization test (HIST) has historically been performed to guarantee the safety of acellular per­tussis vaccine batches. Non-compliance of batches is primarily associated with the presence of low levels of pertussis toxin (PTx). Because of ethical, standardization and scientific reasons, a variety of alternative in vitro approaches have been studied to replace the lethal HIST. A broadly applied and partially accepted method is the CHO cell clustering test, which is based on the clustered growth pattern of CHO cells when exposed to minute amounts of PTx. One of the major hurdles for global application of the CHO clustering test is the manual assessment of the clusters, which is associated with suboptimal reproducibility of test outcomes and is time-consuming. Here, various parameters of CHO cell nuclei were evaluated in search for a reliable, objective read-out parameter. We demonstrate that the distance between each nucleus and its nearest neighbor (3N method) is the most suitable parameter to assess clustered cell growth. This method detects 2.8 mIU PTx/mL and thereby complies with the requirement set for the sensitivity of the CHO clustering test based on visual reading. In commercial acellular pertussis vaccines spiked with PTx, the method detects 45 mIU/mL PTx, which is substantially lower than the 181-725 mIU/mL PTx detected by visual interpretation. The 3N method thus allows objective and sensitive assessment of CHO clustering and thereby encourages broad and global implementation of the in vitro test as an alternative to the HIST.

Reporter cell lines for detection of pertussis toxin in acellular pertussis vaccines as a functional animal-free alternative to the in vivo histamine sensitization test.

Detoxified pertussis toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual pertussis toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST.

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation.

The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and in vivo potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest in vivo potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells in vitro were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the lgmA-C genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring in vitro testing of vaccine quality a step closer.

The cAMP assay: a functional in vitro alternative to the in vivo Histamine Sensitization test.

Safety requirements stipulate the performance of the in vivo Histamine Sensitization (HS) test for quality control of acellular pertussis (aP) vaccines. For reasons of reproducibility and animal welfare concern, an in vitro assay was developed. The assay reflects the mechanism of histamine sensitization and is based on cAMP production in A10 cells to residual pertussis toxin (PT). We showed that PT induces cAMP levels in a dose-dependent manner while the sensitivity of the assay equals the sensitivity of the HS test. Neither the individual components nor the combination vaccine DTaP-IP did affect the assay. The cAMP assay meets the criteria for specificity and sensitivity and therefore might be a promising candidate to replace the HS test.