30. 針刺麻酔に関する文献的ならびに基礎的研究(第519回千葉医学会例会 第8回佐藤外科例会)

<p>Cytokine response in lung homogenates, BALF and plasma of WT and <i>Trem-2</i>^<i>-/-</i> mice during experimental melioidosis.</p

Impact of pDC on vaginal HSV-2 infection.

<p>CLEC4C-DTR Tg mice were injected subcutaneously with Depo-Provera or medroxyprogesterone acetate 7 days before vaginal HSV-2 infection. Mice were injected i.p. with PBS or DT 24 h before infection and every other day after. (A) Dotplots show pDC frequencies in lumbar lymph nodes of PBS or DT-treated mice at the time of infection. HSV-2 titers (B) and IFN-α levels (C) in vaginal/cervical tissues on day 2 p.i. after inoculation with 1×10³ pfu. (D) Control or pDC-depleted mice were infected with 2×10³ or 1×10⁴ pfu of HSV-2 and survival was monitored for 15 days. Data are combined from or are representative of two independent experiments. Not significant (NS).</p

Contribution of pDC to virus-specific CD8 T cell responses after local HSV-1 infection.

<p>Control (PBS) and pDC-depleted (DT) mice were infected in the footpad with HSV-1. Draining popliteal lymph nodes (DLN) and spleens were analyzed on day 7 p.i. (A) Dotplots show pDC frequencies in popliteal lymph nodes of PBS or DT-treated mice at the time of infection. (B) pDC numbers in DLN of PBS or DT-treated mice on day 7 p.i. (C) Total numbers of CD8 and CD4 T cells in DLN. (D) Antigen-specific lysis using DLN cells as effectors and HSV gB peptide-pulsed or unpulsed EL4 cells as targets was assessed in standard 4 h ⁵¹Cr release assays. (E, F) Frequencies of CD8 T cells producing IFN-γ in DLN and spleens after restimulation with HSV gB peptide. Data are combined from or are representative of two independent experiments.</p

TLR3-expressing cells promote CD8 T cell responses to local HSV-1 infection.

<p>WT and TLR3^−/− mice were infected in the footpad with HSV-1 (1×10⁵ pfu). Total CD4 (A) and CD8 (B) T cells were analyzed in DLN on day 7 p.i. (C) Numbers of IFN-γ-producing CD8 T cells in DLN on day 7 p.i. after restimulation with HSV gB peptide. Data are combined from two independent experiments. Statistical significance is indicated by <i>p</i> values.</p

pDC produce IFN-I and activate NK cells during systemic HSV-2 infection.

<p>CLEC4C-DTR Tg mice were depleted (DT) or not (PBS) of pDC then infected i.v. with HSV-2 (5×10⁶ pfu). (A) Serum IFN-α, IFN-γ and IL-12p70 levels were measured 8 h p.i. (B) Serum IFN-α levels at 12 h p.i. in control and pDC-depleted mice. Frequencies of IFN-γ-producing NK cells (NK1.1⁺CD3⁻) (C, D) and CD107a⁺ NK cells (E) in spleens were determined 12 h p.i. (F) PBS and DT-treated mice were infected i.v. with different doses of HSV-2. Survival was monitored for 15 days. Data are representative of two independent experiments. Statistical significance is indicated by <i>p</i> values. Not significant (NS).</p

pDC promote antiviral responses during systemic HSV-1 infection.

<p>Control (PBS) and pDC-depleted (DT) mice were infected i.v. with HSV-1 (1×10⁷ pfu). Serum IFN-α (A) and IFN-γ (B) were measured at 6 and 12 h p.i. (C) CD69 expression on NK cells (NK1.1⁺CD3⁻) from spleens of PBS or DT-treated mice 12 h p.i. Frequencies of CD107a⁺ (D) and IFN-γ-producing NK cells (E, F) in spleens of HSV-1 infected mice 12 h p.i. (G) Frequencies of IFN-γ-producing NKT cells (NK1.1⁺CD3⁺) in spleens of HSV-1 infected mice 12 h p.i. (H) Frequencies of IFN-γ-producing CD8 T cells in spleens on day 7 p.i. after restimulation with HSV gB peptide. (I) CFSE-labeled CD8 T cells from gBT-I mice were injected i.v. into CLEC4C-DTR Tg mice. Mice were depleted or not of pDC and infected i.v. with HSV-1 (1×10⁷ pfu). Spleens were analyzed on day 3 p.i. for numbers of transferred CD8 T cells, CFSE dilution and CD25 levels. Dotplots and histograms are representative of four mice per group. Data are representative of one (I) or two-three independent experiments (A–H). Statistical significance is indicated by <i>p</i> values. Not significant (NS).</p

SeV replication is enhanced in MDA5^−/− mice.

<p>WT and MDA5^−/− mice infected with 200K pfu SeV were assessed for A) SeV replication by IF detection of SeV antigens and by real time PCR analysis of B) SeV genome and C) SeV N gene expression. N = 4, error bars refer to SEM, * P<0.05; ** P<0.005.</p

MDA5 is required for sustained expression of cytokines in response to SeV infection.

<p>Real time PCR analysis of whole lung homogenates obtained from WT and MDA5^−/− mice infected with 200K pfu SeV for expression levels of A) <i>Ifn-α2</i>, B) <i>Ifn-β</i>, C) <i>Ifn-γ</i>, D) <i>Il-28b</i>, E) <i>Tnf-α</i>, F) <i>Il-1β</i>, G) <i>Il-6 and</i> H) <i>Il-10</i> mRNA. N = 4, error bars refer to SEM, * P<0.05, ** P<0.00001.</p

MDA5 deficiency leads to increased MNV titers <i>in vitro</i>.

<p>Bone marrow-derived dendritic cells from wild type (WT), MDA5−/−, or TLR3−/− mice were inoculated with MNV at an MOI of 5 (A) or 0.05 (B) or pre-treated with 20 U IFNα and then inoculated with an MOI of 0.05 (C). Viral titers were done at 6 hour time-points for each sample and statistical significance was determined using student's t test. There was no significant difference between WT and TLR3−/− titers, statistical significance is marked between WT and MDA5−/− titers where * = p<0.05. Data shown is the average of four independent experiments (A and B) or three independent experiments (C). In (D) supernatants from WT BMDC infected at MOI 0.05 were harvested at various time-points and tested for IFNβ by ELISA. Data shown is the average of three independent experiments.</p

Infection with SeV results in induction of antiviral sensor expression.

<p>A) Analysis of <i>Mda5</i> and <i>Rig-I</i> mRNA expression in WT mice during the acute SeV infection period as determined by real-time PCR analysis. B) Micrographs taken of lung sections obtained from WT and MDA5^−/− mice infected with 200K pfu SeV and stained for MDA5 expression. N = 4, error bars refer to SEM, * P<0.05.</p