Upregulation of co-stimulatory cell-surface proteins by PorB is partially TLR2 dependent.

<p>WT and TLR2 KO BMDMs were stimulated in culture with PorB, Pam3CSK4 or LOS, then stained for markers of activation and examined by flow cytometry. In WT cells, PorB increased surface expression of CD14, CD40, CD54, CD69 and CD86 as compared to unstimulated cells. Knockout cells had decreased expression of CD14, CD40 and CD86, but still had higher expression of CD54 and CD69 when compared to basal levels. In the latter two cases, the TLR2 ligand Pam3CSK4 had no effect on TLR2 -/- cells. Histograms are from one representative sample of at least 3. Data represents one of two experiments.</p

PorB induction of pro-inflammatory cytokines in macrophages is MAPK dependent.

<p>(a,b) PorB potently induces IL-6 and TNFα. Supernatants were collected from WT BMDM stimulated with PorB, Pam3CSK4 or <i>N. meningitidis</i> LOS at multiple time points. At 12, 24 and 48 hours post-stimulation levels IL-6 (a) of TNFα (b) were significantly higher than unstimulated cells. (c,d) Induction of inflammatory cytokines by PorB is inhibited by blocking MAPK. PorB, Pam3CSK4 or LOS were added to cultures of WT mouse BMDM. Cells were inhibited with the carrier DMSO, or MAPK pathway inhibitors, and production of inflammatory cytokines was measured. All inhibitors tested showed significant reductions in the stimulatory activity of PorB on the production of IL-6 (c) and/or TNFα (d). U: UO126, an inhibitor of Erk1/2 by inhibiting MEK1/2. SB: SB203580, a direct inhibitor of p38. SP: SP600125, a direct inhibitor of jnk. Data represents one of three experiments.</p

Increases in antigen-specific IgG by PorB is dependent on TLR2 and MyD88.

<p>(a) Concentration of IgG antibody to Ova in C57Bl/6 mice as measured by ELISA in serum on from day 42. WT C57Bl/6, TLR2 ^-/- and MyD88 ^-/- mice were vaccinated on days 0, 14 and 28 with Ova, Ova + PorB or sham (PBS). Vaccines that included PorB showed significantly less elevation in IgG over vaccines that did not include PorB in TLR2 ^-/- mice, and no detectable effect in MyD88 ^-/- mice. None of the mice had detectable antibodies to Ova prior to vaccination (data not shown). (b) OD of specific subtypes of anti-Ova IgG at a 1:50 dilution of serum as measured by ELISA. In WT mice, IgG1 and IgG2b were the dominant subtypes indicative of a Th2-type response. These in turn decreased in the TLR2 ^-/- mice, consistent with the total IgG data. Data represents one of two experiments. * p<0.05.</p

IL-1β induction by PorB requires ATP to activate the inflammasome.

<p>WT BMDMs were stimulated with PorB, LPS, TNFα, or Pam₃CSK₄ at the indicated concentrations for 5 hours. After 5 hours, ATP was added to half the cells for 30 minutes. Supernatants were collected and analyzed by IL-1β ELISA. IL-1β release was not observed for PorB, LPS or Pam₃CSK₄ in the absence of ATP. In the presence of ATP, significantly increased IL-1β release was observed for all three TLR ligands (p<0.05). </p

Supplemental Material, sj-docx-1-afp-10.1177_19253621221124800 - Postmortem Genetic Testing Is an Increasingly Utilized Tool in Death Investigation

Supplemental Material, sj-docx-1-afp-10.1177_19253621221124800 for Postmortem Genetic Testing Is an Increasingly Utilized Tool in Death Investigation by Rebecca Latimer, Heather MacLeod, Lisa Dellefave-Castillo, Daniela Macaya and Tara R. Hart in Academic Forensic Pathology</p

Model of chromatin positioning and gene expression.

<p>In the case of the <i>LMNA</i> E161K mutation, two distinct loci on chromosome 13 were displaced to a more intranuclear position (right). We hypothesize that loss of interaction with the lamina (blue) prevents interaction with active chromatin complexes (black) and reduces gene expression.</p

The lamina is intact in <i>LMNA</i> E161K heart and fibroblasts.

<p>(A) Electron microscopy illustrates the electron dense lamina in both the <i>LMNA</i> E161K and <i>LMNA</i> normal hearts, and shows no appreciable difference. N = nucleus, red arrows indicate nuclear membrane. Scale bar  = 2 µm. (B) The LINC complex proteins localize normally in <i>LMNA</i> E161K mutant heart. Sections from <i>LMNA</i> E161K mutant and <i>LMNA</i> normal heart were analyzed by immunofluorescence microscopy using antibodies for lamin A/C (green), nesprin-1, emerin and SUN1 (red). DAPI is shown in blue. Scale bar  = 10 µm. (C) Lamin A/C (green) localization was determined using immunofluorescence microscopy in <i>LMNA</i> E161K mutant and <i>LMNA</i> normal fibroblasts. DAPI shown in blue. Scale bar  = 10 µm.</p

Genes misexpressed in both LMNA mutant heart and fibroblasts.

<p>Genes misexpressed in both LMNA mutant heart and fibroblasts.</p

Two gene clusters on chromosome 13 are displaced from the nuclear periphery in <i>LMNA</i> E161K cells.

<p>Gene expression profiling identified gene clusters that were misexpressed in <i>LMNA</i> E161K. Two clusters from chromosome 13, referred to as 13A and 13B were studied because they contain genes important for striated muscle function. (A) Cluster 13A contains <i>LMO7</i> which encodes a nuclear membrane associated emerin-interacting protein. Cluster 13B contains <i>MBNL2.</i> The intranuclear position of Cluster 13A (red, top) and Cluster 13B (green, bottom) is shown in <i>LMNA</i>-normal nuclei and <i>LMNA</i>-mutant nuclei. Anti-Lamin B-1 (αLMNB1) is shown in blue. (B) The nuclear position of Cluster A was displaced away from the nuclear periphery in <i>LMNA</i> mutant versus normal (n = 98 control nuclei and n = 64 E161K nuclei), (*p = 0.0001)(top). Similarly, the nuclear position of Cluster B was displaced towards the nuclear center in <i>LMNA</i> E161K mutant versus <i>LMNA</i> normal nuclei (n = 106 control nuclei and n = 66 for E161K nuclei) (*p = 0.02) (bottom). (C) The nuclear position of the <i>ACTB</i> gene encoding β-actin was examined as a control genomic locus and did not differ between mutant and normal. Anti-Lamin B-1 (αLMNB1) is shown in green and DAPI staining in blue. Scale bar = 10 µm. (D) There was no significant difference between the localization of the ACTB locus in E161K <i>LMNA</i> mutant versus <i>LMNA</i> normal nuclei.</p

Genomic clusters of misexpressed genes.

<p>Shown are genes that are misexpressed in the LMNA mutant heart that are colocalized in the same genomic interval. The chromosome position is shown on the left. The two genes within each interval are indicated in the subsequent columns.</p