Treatment of microglia with NCM and tuftsin induced the release of immunomodulatory factors.

<p>To characterize the factors released from microglia under EAE-like conditions, microglia were treated for 10 hours with NCM isolated from neurons undergoing excitotoxic injury in the presence or absence of tuftsin. Concentrated conditioned media were analyzed by multidimensional chromatography and tandem mass spectrometry. Partial list of the secreted proteins is shown. Spectral counts (MS/MS) of proteins identified from the media of microglia treated with NCM alone (MgCM) or a combination of NCM and 100 µg/ml of tuftsin (MgCM+Tuf) for 10 hours are listed in the respected columns. Ratios derived from spectral counts were used to predict the trend of differential expression.</p

Adoptive transfer of tuftsin and NCM modulated T cells on day 14 ameliorates established EAE.

<p>Wild-type (WT) mice were injected with MOG_35–55 peptide in CFA and pertussis toxin to induce EAE. Osmotic pumps filled with PBS or 500 µM tuftsin in PBS were implanted in the back of the mice on day 1 after MOG immunization. 5×10⁶ T cells, modulated as follows: tuftsin/NCM-treated T cells; tuftsin-only treated T cells; NCM-only treated T cells, were injected intravenously into recipient C57BL/6 mice immunized with MOG 14 days after the onset of disease (arrow indicates timepoint when T cell transfer was performed) and the disease severity scored as described in Methods. (The experiment was repeated twice. Cumulative data are provided; total number of animals tested, n = 12/PBS-infused mice; n = 12/tuftsin-infused mice; n = 12/mice adoptively transferred with tuftsin plus NCM-modulated T cells; n = 5/mice adoptively transferred with tuftsin-only treated T cells; n = 5/mice adoptively transferred with NCM-only treated T cells). Statistical analysis was performed, as described in the Methods. All time points between tuftsin/NCM-treated T cells and PBS control were found to be significant (p<0.001) except for days 14–15, 34 and 41–44). All the timepoints between tuftsin alone and PBS were significant (p<0.001). No difference was evident between tuftsin and tuftsin/NCM-treated T cells.</p

Treatment of microglia with tuftsin and excitotoxic media causes an M2 shift.

<p>Quantitative RT-PCR was performed to analyze changes in gene expression of the M1 marker TNFα (<b>A</b>) and the M2 marker IL10 (<b>B</b>) in response to tuftsin and NCM treatment for 10 hours. Primary microglia were treated with increasing concentrations of tuftsin or tuftsin and NCM prepared from primary cortical neurons exposed to 100 µM glutamate overnight. (<b>C</b>) The ratio of the fold change of IL10 (M2) to the fold change of TNFα (M1). n = 3, *, p<0.5; **, p<0.01; ***, p<0.001. In (<b>A</b>) and (<b>B</b>) the comparisons are between tuftsin and tuftsin/NCM at each concentration. In (<b>C</b>) the comparisons are between NCM and tuftsin/NCM.</p

Cytokine production from direct or indirect exposure of T cells to tuftsin.

<p>(<b>A</b>) ELISA assays were performed to quantitate the proinflammatory cytokine TNFα and the anti-inflammatory cytokine IL10 as produced by splenic T cells exposed directly to varied concentrations of tuftsin, as described in Methods. n = 4, *, p<0.5; **, p<0.01; ***, p<0.001. When ELISA assays were performed to quantitate pro-inflammatory cytokine TNFα (<b>B</b>) and anti-inflammatory cytokine IL10 (<b>C</b>) production by T cells after treatment with microglial-conditioned medium (MgCM). The microglia were challenged either with tuftsin or with tuftsin and conditioned medium from neurons (NCM) that had been exposed to glutamate. n = 4, *, p<0.5; **, p<0.01; ***, p<0.001.</p

Tuftsin infusion induces the expression of Th2 and Treg cell markers.

<p>Western blot analyses were performed on mouse spinal cord extracts at different timepoints following EAE induction to examine protein expression in the presence or absence of 500 µM tuftsin infusion. (<b>A</b>) Phosphorylation of STAT1 and the levels of the anti-inflammatory cytokines IL10 and IL4 were assessed in extracts from tuftsin-infused wt mice subjected to EAE. Equal loading of protein was confirmed by pan-STAT1 blotting. (<b>B</b>) Levels of the Treg transcription factor FoxP3 and the Treg cell surface marker CD25. Equal loading of protein was confirmed by actin blotting. (Western blots were repeated three times on mouse spinal cord samples from three separate EAE experiments, n = 3).</p

PCR array results indicating changes in mRNA levels of T cells after tuftsin treatment.

<p>T cells were treated with MgCM that was isolated from microglia treated with NCM alone, 100 µg/ml of tuftsin alone, or a combination of NCM+100 µg/ml tuftsin for 10 hours. After 3 days, mRNA was isolated from T cells and was reverse-transcribed and used to probe a PCR array kit of inflammatory cytokines and their receptors to determine the subtype of T cells present as a result of treatment, as described in Methods. n = 2.</p

PCR array results indicating changes in mRNA levels of T cells after tuftsin treatment.

<p>T cells were treated with MgCM that was isolated from microglia treated with NCM alone, 100 µg/ml of tuftsin alone, or a combination of NCM+100 µg/ml tuftsin for 10 hours. After 3 days, mRNA was isolated from T cells and was reverse-transcribed and used to probe a PCR array kit of inflammatory cytokines and their receptors to determine the subtype of T cells present as a result of treatment, as described in Methods. n = 2.</p

Detecting the Immune System Response of a 500 Year-Old Inca Mummy

<div><p>Disease detection in historical samples currently relies on DNA extraction and amplification, or immunoassays. These techniques only establish pathogen presence rather than active disease. We report the first use of shotgun proteomics to detect the protein expression profile of buccal swabs and cloth samples from two 500-year-old Andean mummies. The profile of one of the mummies is consistent with immune system response to severe pulmonary bacterial infection at the time of death. Presence of a probably pathogenic <em>Mycobacterium sp</em>. in one buccal swab was confirmed by DNA amplification, sequencing, and phylogenetic analyses. Our study provides positive evidence of active pathogenic infection in an ancient sample for the first time. The protocol introduced here is less susceptible to contamination than DNA-based or immunoassay-based studies. In scarce forensic samples, shotgun proteomics narrows the range of pathogens to detect using DNA assays, reducing cost. This analytical technique can be broadly applied for detecting infection in ancient samples to answer questions on the historical ecology of specific pathogens, as well as in medico-legal cases when active pathogenic infection is suspected.</p> </div

Maximum likelihood phylogeny.

<p>Maximum likelihood phylogeny and bootstrap support values based on 1000 pseudoreplicates of the alignment of <i>hsp65</i> gene nucleotide sequences. Sequences generated from the Maiden’s swab samples are shown in bold, larger font, and marked with an asterisk.</p

Gel electrophoresis from the boy.

<p>Gel electrophoresis (2%) showing the products of PCR amplification from samples from the boy. The results were negative: no amplification bands could be detected. The image was inverted, and contrast was increased to facilitate detection of bands. No other image treatment was performed. Lanes 1 and 2 were amplified with <i>M. tuberculosis</i>-specific primers, lanes 3 and 4 with the <i>Mycobacterium</i>-specific primers, and lanes 5, 6 and 7 were negative controls.</p