Levels of Anti-TMV IgG Subclass in Human Serum.

<p>Sera were obtained from groups of smokers and non-tobacco users (n = 20/group). Serum anti-TMV IgG subclass levels were measured by a customized sandwich ELISA assay. The serum dilution factor for anti-TMV IgG1/IgG3/IgG4 was 1∶100 and for anti-TMV-IgG2 was 1∶10. Data represent two to three independent experiments with double wells per subject. Results are expressed as O.D. values (mean ± SE); <i>p</i>-value, ^a Student’s <i>t</i> test, *<i>p</i> < 0.05.</p

Effects of tobacco-smoke and smokeless-tobacco exposure on serum anti-Tobacco Mosaic Virus (TMV) IgG.

<p>Sera were obtained from groups of smoke tobacco users (smokers), smokeless tobacco users (tobacco chewers) and non-tobacco users (n  =  20/group). Serum anti-TMV IgG levels were measured by a customized sandwich ELISA assay. The serum dilution factor was 1∶100 and the data is expressed as O.D. value (mean ± SE). All data are representative of two to three assays with similar results. (A) Serum anti-TMV IgG in whole study population; (B) Ethnic characteristics of the study population; (C) Serum anti-TMV IgG in African American study population; and (D) Serum anti-TMV IgG in Caucasian study population. <i>p</i>-value, Student’s <i>t</i> test, *<i>p</i> < 0.05.</p

Humans Have Antibodies against a Plant Virus: Evidence from Tobacco Mosaic Virus

<div><p>Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog – like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson’s disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson’s disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored.</p> </div

Cross reactivity between TMV and TOMM40L.

<p>(A) Sequence homology comparison among TMV-Vulgare coat protein. Basic Local Alignment Search Tool (BLAST) indicated the regions of local sequence similarity (color red) among TMV and TOMM40L gene and TOMM40L peptide; and sequence similarity (color green) between TOMM40L gene and peptide. (B) Cross-reactivity of anti-TMV antibodies and TOMM40L peptide determined by tobacco mosaic virus PathoScreen ELISA kit. Data are representative of two independent experiments with similar results. Results were expressed as O.D. values (mean ± SE, triple wells per group). Tom40  =  TOMM40L peptide, Mito  =  crude mitochondria pellet, TMV (+)  =  TMV-infected tobacco leaf, TMV (-)  =  TMV-negative alstroemeria leaf, and control  =  ELISA assay buffer.</p

Effects of tobacco-smoke and smokeless-tobacco exposure on serum anti-TMV IgA, IgM, IgD and IgE.

<p>Sera were obtained from groups of smokers, smokeless tobacco users and non-tobacco users (n = 20/group). Serum anti-TMV IgA, IgM, IgD, and IgE levels were measured by a customized sandwich ELISA assay. The serum dilution factors were 1∶50 for anti-TMV IgA and IgM and 1∶5 for anti-TMV IgD and IgE. Data is expressed as O.D. values (mean ± SE). All data are representative of two to three assays with similar results. (A) Serum anti-TMV IgA in whole study population; (B) Serum anti-TMV IgM in whole study population; (C) Serum anti-TMV IgD in whole study population; (D) Serum anti-TMV IgG in whole study population. <i>p</i>-value, ANOVA, *<i>p</i> < 0.05.</p

The KD ameliorates EAE-mediated CNS-inflammation and oxidative stress.

<p>Comparison of the frequencies of (A) CD4⁺/CD8⁺ T cells, (B) CD11b⁺/CD45⁺ cells on gated lymphocyte populations in EAE with and without KD treatment. (C) Frequencies of CD4⁺CD25⁺ Foxp3⁺ regulatory T cells. The KD suppressed CNS cellular infiltration and myelin-reactive T cell responses in EAE. Groups of mice (receiving standard diet [SD] and KD diet) were immunized with MOG/CFA/PT, and sacrificed between day 16–19 p.i. Lymph node, spleen and CNS cells were isolated. (D) Lymphoid (LN) or CNS cells were re-stimulated with MOG_35–55 peptide overnight and IFN-γ- and IL-17-expressing CD4⁺ T cells were measured by intracellular staining. The number seen in the box indicated the average percentage of individual markers on the cells. (E) Summary data of the percentage of individual marker in the cells (LN <i>vs.</i> CNS) in SD-treated EAE mice or KD-treated EAE mice. Data are representative of two independent experiments (n = 4–8/group). P values, Student’s <i>t</i>-test; *, <i>p</i><0.05. (E) Visualization and quantification of brain inflammation by <i>in vivo</i> bioluminescence imaging. This is achieved by imaging reactive oxygen species (ROS) levels in the brain. Bioluminescent images in live mice were captured during a 1 min acquisition time using the Xenogen IVIS system at several time points after injection of 27 mg/kg dihydroethidium. Representative images of ROS seen in the left panel were captured in naïve mice and EAE mice with and without KD treatment. All experiments were conducted on groups of mice (n = 4–8) between days 14–19 p.i. Data are representative of two independent experiments (mean and SEM). P values, one way ANOVA; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Structural brain changes in EAE mice with and without KD treatment.

<p>(A) T2-weighted images were obtained with a 7T MR scanner during the peak stage of motor disability. EAE mice showed abnormal signal intensities adjacent to the lateral ventricles. Arrows indicate focal lesions on T2-weighted imaging. In contrast, lesion volumes were significantly reduced in KD-fed EAE mice (N = 4–8). (B) In contrast to the observed hippocampal volume loss in EAE mice at 30 days post immunization, neither naïve mice nor KD-fed EAE mice showed changes in hippocampal volume.</p

Effects of the ketogenic diet on motor disability and memory deficit in EAE mice.

<p>(A) Starting from day 17 post immunization (p.i.), KD-fed EAE mice (n = 11) showed significantly less motor disability than SD-fed EAE mice (**, <i>p<</i>0.01, Mann-Whitney <i>U</i> test). In KD- fed and SD- fed EAE mice, the mean clinical scores (1.9±0.20 vs 2.7±0.35 and 0.43±0.12 vs 1.0±0.15) were measured at 17 days and 25 days p.i., respectively. (B, C) Restoration of spatial learning and memory by the KD in EAE mice. MWM testing was conducted in groups of EAE mice either prior to the occurrence of motor deficits (before 10 days p.i., left panel, B) or from 30 days to 35 days p.i. (right panel, C). The graph illustrates average latencies to finding the submerged escape platform. Each group consisted of 5–8 mice. Values represent mean±SEM. One way ANOVA followed by Tukey test. (D) Change in the synaptic plasticity at 13–16 days p.i. with or without the KD. EAE mice exerted impairment in LTP after high-frequency stimulation (10 slices form 5 mice). Both the naïve and KD-fed EAE mice demonstrated intact LTP induction and maintenance (9 slices from 4 mice). (E) Change in synaptic plasticity at 25–35 days p.i. with or without the KD. Neither the naïve nor KD-fed EAE mice showed changes in LTP maintenance (10 slices from 5 mice). The dotted line indicates the baseline field potential amplitude. Synaptic plasticity seen during peak and mild stages of motor disability are depicted in the left and right panels, respectively.</p

Table_8_Whole–genome sequencing, annotation, and biological characterization of a novel Siphoviridae phage against multi–drug resistant Propionibacterium acne.DOCX

Antibiotics-resistant Propionibacterium acne (P. acne) causes severe acne vulgaris, serious public health, and psychological threat. A new lytic bacteriophage (phage), φPaP11-13, infecting P. acne, was isolated from the sewage management center of Xinqiao Hospital. It can form transparent plaque with diameters of 1.0 ~ 5.0 mm on the double-layer agar plate, indicating a robust lytic ability against its host. Transmission electron microscopy (TEM) showed that φPaP11-13 belonged to the Siphoviridae family (head diameter 60 ± 4.5 nm, tail length 170 ± 6.4 nm, tail width 14 ± 2.4 nm). The one-step growth curve showed the incubation period was 5 h, and the burst size was 26 PFU (plaque-forming unit)/cell. Moreover, it exhibited tolerance over a broad range of pH and temperature ranges but was utterly inactivated by ultraviolet (UV) irradiation for 1 h. The whole-genome sequencing results revealed φPaP11-13 had a linear dsDNA with 29,648 bp length. The G/C content was 54.08%. Non-coding RNA genes and virulence factors were not found. Forty five open reading frames (ORFs) were identified after online annotation. This study reports a novel P. acne phage φPaP11-13, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of φPaP11-13 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling antibiotics-resistant P. acne-induced severe acne vulgaris.</p

Table_7_Whole–genome sequencing, annotation, and biological characterization of a novel Siphoviridae phage against multi–drug resistant Propionibacterium acne.DOCX

Antibiotics-resistant Propionibacterium acne (P. acne) causes severe acne vulgaris, serious public health, and psychological threat. A new lytic bacteriophage (phage), φPaP11-13, infecting P. acne, was isolated from the sewage management center of Xinqiao Hospital. It can form transparent plaque with diameters of 1.0 ~ 5.0 mm on the double-layer agar plate, indicating a robust lytic ability against its host. Transmission electron microscopy (TEM) showed that φPaP11-13 belonged to the Siphoviridae family (head diameter 60 ± 4.5 nm, tail length 170 ± 6.4 nm, tail width 14 ± 2.4 nm). The one-step growth curve showed the incubation period was 5 h, and the burst size was 26 PFU (plaque-forming unit)/cell. Moreover, it exhibited tolerance over a broad range of pH and temperature ranges but was utterly inactivated by ultraviolet (UV) irradiation for 1 h. The whole-genome sequencing results revealed φPaP11-13 had a linear dsDNA with 29,648 bp length. The G/C content was 54.08%. Non-coding RNA genes and virulence factors were not found. Forty five open reading frames (ORFs) were identified after online annotation. This study reports a novel P. acne phage φPaP11-13, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of φPaP11-13 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling antibiotics-resistant P. acne-induced severe acne vulgaris.</p