The Role of Innate Immune Responses in the Outcome of Interspecies Competition for Colonization of Mucosal Surfaces Lysenko ES.et al PLoS Pathog volume 1, 生科四甲 張耀文

Introduction For many microorganisms, including some that have the potential to behave as pathogens, their primary interaction with a host is through stable colonization of mucosal surfaces.

the gram-positive the gram-positive Streptococcus pneumoniae (S.pneumoniae) Streptococcus pneumoniae (S.pneumoniae) and the gram negative and the gram negative Haemophilus influenzae (H.influenzae). Haemophilus influenzae (H.influenzae). The purpose of this study was to examine the interaction between these two species in vivo during experimental colonization.

縮寫 H&E, hematoxylin and eosin; H&E, hematoxylin and eosin; LPS, lipopolysaccharide; LPS, lipopolysaccharide; mAb, monoclonal antibody; mAb, monoclonal antibody; MIP-2, macrophage inhibitory protein 2; MIP-2, macrophage inhibitory protein 2; PEC, peritoneal exudate cell; PEC, peritoneal exudate cell; SCID, severe combined immunodeficiency SCID, severe combined immunodeficiency

Materials and Methods Bacterial strains and culture conditions. Hi636 (a type b capsule-expressing, spontaneously streptomycin-resistant mutant of H. influenzae strain Eagan ) Sp1121 (a type 23F capsule-expressing).

老鼠的實驗品種 Six-week-old C.B-17/lcrCrlBR (BALB/c SCID) C3H/HeOuJ ( 有免疫能力的正常老鼠 )

實驗主要流程圖 觀察 H. influenzae 和 S.pneumoniae 在 SCID mice 和 C3H mice 中的 生長情況 測定 MIP-2 的濃度 補體和嗜中性球的消耗 對於 co-colonization 的影響

細菌的培養 每組至少五隻老鼠，在內鼻接種 mid-log phase 的 1.H. influenzae 1.H. influenzae 2.S. pneumoniae 2.S. pneumoniae 3. 都接種 3. 都接種 24 小時後插管，倒入 PBS ，收集洗滌液。將 洗滌液連續稀釋後培養在含有抗生素的培 養基上。

Figure 1. Colonization of BALB/c SCID mice by H. influenzae Strain Hi636 and S. pneumoniae Strain Sp1121 In SCID mice

抗體螢光染色 為了抗體螢光染色，利用 Tissue-Tek O.C.T. 冷凍 包埋。 為了抗體螢光染色，利用 Tissue-Tek O.C.T. 冷凍 包埋。 5um 一小片段  air dried  fixed in acetone at 4 o C. 5um 一小片段  air dried  fixed in acetone at 4 o C. 培養 在包含羊血清的 PBS for 30 mins 培養 在包含羊血清的 PBS for 30 mins 加入已經稀釋 1000 倍的主要抗體 anti–type b H. influenzae 或 anti-type 23F S. pneumoniae 培養 在室溫下 60 分鐘。 加入已經稀釋 1000 倍的主要抗體 anti–type b H. influenzae 或 anti-type 23F S. pneumoniae 培養 在室溫下 60 分鐘。 之後加入次要抗體 ( 在室溫下培養 60 分鐘。再用顯微鏡觀 察螢光。 之後加入次要抗體 (Texas Red–labeled goat anti- rabbit IgG) 在室溫下培養 60 分鐘。再用顯微鏡觀 察螢光。

Figure 2. Immunofluorescence Showing Co-localization of H. influenzae and S. pneumoniae in the Murine Nasopharynx immunofluorescence

Figure 3. Competition between Species during Co-Colonization of Immunocompeten Mice In C3H mice

Figure 4. H&E-Stained Parasagittal Sections Showing the Luminal Space between Two Adjacent Nasal Turbinates

Figure 5. The Effect of Co-Colonization on the Concentration of MIP-2 in Upper Respiratory Tract Lavage Fluid Measurement of MIP-2 concentration.

Figure 6. The Effect of Depletion of Neutrophil-Like Cells on Competition between Species during Co-Colonization

Figure 7. The Effect of Complement Depletion on Competition between Species during Co-Colonization

Conclusion Clearance of S. pneumoniae required both complement and the recruitment of neutrophil-like cells to the mucosal surface. Stimulation of neutrophil-like cells with bacterial components of H. influenzae was required for efficient clearance of S. pneumoniae.

The End Thank You

Hematoxyline : 5 分鐘 沖掉 Hematoxyline D.D.water 浸潤數秒 PBS 溶液中使組織切片 呈藍色即可（約 5 分鐘） 95 ％ alcohol : 30 秒 eosin : 30 秒 95 ％ alcohol 浸潤數秒 100 ％ alcohol 浸潤數秒 xylene I:5 分鐘 xylene II:5 分鐘 xylene III: 浸泡直到用膠封片 H & E 染色