Mechanisms of cell injury induced by inhaled molybdenum trioxide nanoparticles in Golden Syrian Hamsters.

Molybdenum trioxide nanoparticles (MoO3 NPs) are extensively used in the biomedical, agricultural, and engineering fields that may increase exposure and adverse health effects to the human population. The purpose of this study is to evaluate a possible molecular mechanism leading to cell damage and death following pulmonary exposure to inhaled MoO3 NPs. Animals were separated into four groups: two control groups exposed to room air or aerosolized water and two treated groups exposed to aerosolized MoO3 NPs with a concentration of 5 mg/m3 NPs (4 h/day for eight days) and given a one-day (T-1) or seven-day (T-7) recovery period post exposure. Pulmonary toxicity was evaluated with total and differential cell counts. Increases were seen in total cell numbers, neutrophils, and multinucleated macrophages in the T-1 group, with increases in lymphocytes in the T-7 group (*P < 0.05). To evaluate the mechanism of toxicity, protein levels of Beclin-1, light chain 3 (LC3)-I/II, P-62, cathepsin B, NLRP3, ASC, caspase-1, interleukin (IL)-1ß, and tumor necrosis factor-α (TNF-α) were assessed in lung tissue. Immunoblot analyses indicated 1.4- and 1.8-fold increases in Beclin-1 in treated groups (T-1 and T-7, respectively, *P < 0.05), but no change in protein levels of LC3-I/II in either treated group. The levels of cathepsin B were 2.8- and 2.3-fold higher in treated lungs (T-1 and T-7, respectively, *P < 0.05), the levels of NLRP3 had a fold increase of 2.5 and 3.6 (T-1 *P < 0.05, T-7 **P < 0.01, respectively), and the levels of caspase-1 indicated a 3.8- and 3.0-fold increase in treated lungs (T-1 and T-7, respectively, *P < 0.05). Morphological changes were studied using light and electron microscopy showing alterations to airway epithelium and the alveoli, along with particle internalization in macrophages. The results from this study may indicate that inhalation exposure to MoO3 NPs may interrupt the autophagic flux and induce cytotoxicity and lung injury through pyroptosis cell death and activation of caspase-1.

Identification of cross-reactive epitopes on the conserved 47-kilodalton antigen of Orientia tsutsugamushi and human serine protease.

Orientia tsutsugamushi is the causative agent of scrub typhus. One of the protein antigens of this species, the conserved 47-kDa protein (HtrA), has been shown to induce an antibody response in patients and can provide protective immunity against live challenge by Orientia in mice. Pepscan experiments identified many peptide epitope clusters in different parts of this protein. The majority of the most reactive epitopes are located at the C terminus of the protein (from amino acid 333 to amino acid 430). Protein sequence analysis revealed that the 47-kDa protein contains a trypsin domain and has sequence homology to human serine protease HtrA1 (hHtrA1). As the 47-kDa protein is a potential vaccine candidate and its ability to induce autoimmunity is a concern, the reactivity of scrub typhus patient sera with purified recombinant 47-kDa and hHtrA1 proteins was tested. A significant percentage (>20%) of scrub typhus patient sera reacted strongly with recombinant hHTRA1 and two of the antigenic polypeptide epitopes in hHtrA1. These findings suggest that the safety of the full-length 47-kDa antigen as a vaccine candidate is a significant issue due to its cross-reactivity with a human protein, which may also contribute to autoimmune responses or enhanced pathology in some scrub typhus patients.

Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus.

We developed a rapid dot-enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available.

Analysis of the cross-reactivity of various 56 kDa recombinant protein antigens with serum samples collected after Orientia tsutsugamushi infection by ELISA.

Orientia tsutsugamushi, the etiologic agent of scrub typhus, has a highly expressed and immunodominant 56-kD outer membrane protein. This protein is one of the leading candidates for diagnosis and vaccine development for scrub typhus. Previous studies using recombinant 56-kD protein (r56s) derived from Karp strain (Kpr56) in a mouse model have shown good homologous protection but only moderate to poor heterologous protection. We evaluated the cross-reactivity of recombinant 56-kD proteins from Karp, Kato, Gilliam, TA763, and three chimeric 56-kD proteins. Not all r56s are equally reactive with strain-specific serum samples. These data provide a first glance of how reactive these r56s are toward the antiserum of different strains and which r56 exhibits the broadest reactivity. A formulation of this combination has the potential to provide broad protection against the heterologous challenge and to be used in a highly sensitive diagnostic assay.

Kinetics and magnitude of antibody responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub typhus patients.

Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.