Expression of p16 in Conjunctival Intraepithelial Neoplasia Does Not Correlate with HPV-Infection

The aim of our study was to identify the frequency of expression of p16INK4a (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN)

TLR9-Dependent and Independent Pathways Drive Activation of the Immune System by Propionibacterium Acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1–2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders

Surfactant protein A detection in large cell carcinoma of the lung

Large cell carcinomas of the lung are undifferentiated malignant epithelial tumors that lack cytologic features of small cell carcinoma, glandular cell carcinoma, or squamous cell differentiation. Lung surfactant protein A (SP-A) is produced by alveolar type 11 cells and Clara cells. Most bronchioloalveolar carcinomas of the lung react positively for SP-A. Positive SP-A staining of large cell carcinoma of the lung could indicate that at least part of these tumors have the same cellular origin or differentiation as bronchioloalveolar carcinoma. The authors determined the SP-A staining of 63 large cell carcinomas of the lung by IHC. In 20 of the 63 (32%), the tumors stained positive for SP-A. This may imply that about one third of large cell carcinomas of the lung have a similar cellular origin or differentiation as bronchioloalveolar carcinoma. The significance of this finding for prognosis and new forms of treatment remains to be determined

Delayed development of <i>P. acnes</i> effects in primed TLR9^−/− mice.

<p>Groups of WT and TLR9^−/− mice (15–20 animals/group) were primed with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated (controls; day 0). <b>A. Hypersensitivity to LPS:</b> At indicated time points after priming the animals were challenged with LPS from <i>S.a.e.</i> (0.01 µg/g b.w.), i.v. One hour and 4 h later, plasma was collected for determination of TNF-α and IFN-γ, respectively. Before challenge with LPS no detectable TNF-α or IFN-γ was found in plasma of <i>P. acnes</i>-treated mice of either one of the groups (not shown). Cumulative data of 3 different experiments are shown. *:p-value<0.05 and ***:p-value<0.001 WT compared to TLR9^−/− mice, and <i>P. acnes</i>-treated WT or <i>P. acnes</i>-treated TLR9^−/− at the indicated time point compared to untreated controls. <b>B. Splenomegaly:</b> Spleens were removed and weighted at indicated time points after treatment. Cumulative data from 4–5 experiments are shown. *:p-value<0.05 and **:p-value<0.01. <b>C. Enhanced resistance to serovar Typhimurium infection is delayed in TLR9^−/− mice:</b> 7 or 21 days after priming, the animals were infected with <i>Salmonella</i> serovar Typhimurium (200 CFU/0.2 ml PBS/i.v.). Bacterial counts in liver of unprimed and primed animals were determined 4 days after infection. One representative experiment of three is shown. *:p-value<0.05 and **:p-value<0.01.</p

LPS sensitivity of <i>P. acnes</i> primed TLR2/TLR4/TLR9^−/− mice after.

<p>Groups of TLR9^−/− and TLR2/TLR4/TLR9^−/− mice were primed with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated. Each mouse was challenged 21 days after priming with a mixture of murine recombinant IL-12 (25 ng) and murine recombinant IL-18 (100 ng). Four hours after challenge, plasma was collected for determination of IFN-γ. Before challenge, no detectable IFN-γ was found in plasma of <i>P. acnes</i>-treated mice of either one of the groups (not shown). One representative experiment of two is shown. *:p-value<0.05 and **:p-value<0.01 as compared to only rIL-12+rIL-18-treated controls.</p

A. Induction of LPS hypersensitivity in mice pretreated with murine recombinant IFN-γ.

<p><b>WT and MyD88^−/− mice were treated with recombinant IFN-γ (5000 </b><b>U/0.2 </b><b>ml) i.v.</b> or remained untreated (only LPS). Eight hours after pretreatment the animals were challenged with LPS <i>S.a.e.</i> (1 µg/g b.w.) i.v. and plasma was collected two hours later for determination of IFN-αβ. One representative experiment of three, with four to five mice is shown. <b>B</b>. Enhanced expression of IL-12Rβ1 and IL-12Rβ2 in MyD88^−/− and WT bone marrow derived macrophages and dendritic cells after stimulation with murine recombinant IFN<b>-</b>γ<b>.</b> 1.5×10⁶ bone marrow derived macrophages (BMM) or dendritic cells (BMDC) were stimulated with murine recombinant IFN-γ (rIFN-γ, 3000 U). After 24 hours, the cells were lysed in guanidinium-thiocyanate solution and lysates were used for RNA preparation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039155#s4" target="_blank">materials and methods</a>. The mRNA levels were analyzed by quantitative real-time PCR. Each value is represented as a relative expression, which is evaluated after setting background expression in controls as arbitrary value  = 1. One representative experiment of two is shown.</p

Absence of hypersensitivity to LPS and splenomegaly in MyD88^−/− mice after <i>P. acnes</i> treatment.

<p>Groups of four to five WT, TLR9^−/− and MyD88^−/− mice were treated with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated (control). 7 and 21 days after priming the animals were challenged with LPS <i>S.a.e.</i> (0.01 µg/g b.w.) i.v. and plasma was collected two hours later for determination of IFN-αβ (<b>A</b>). The spleens were removed and weighted (<b>B</b>). One representative experiment of many is shown. *:p-value<0.05, **:p-value<0.01 and ***:p-value<0.001.</p