International Centre for Diarrheal Disease Research, Bangladesh (icddr,b), Mohakhali, Dhaka 1212, Bangladesh.
BMC Pulm Med. 2013 Apr 16;13:23. doi: 10.1186/1471-2466-13-23.
We earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM).
Healthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method.
MDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05).
The results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007).
我们之前的研究表明，4- 苯丁酸（PB）可以与 1,25- 二羟维生素 D3 协同诱导肺上皮细胞系中的 cathelicidin LL-37 表达。我们旨在评估治疗剂量的 PB 单独或与维生素 D3 联合用于诱导免疫细胞中的 LL-37 表达，并增强单核细胞衍生巨噬细胞（MDM）中的抗分枝杆菌活性。
健康志愿者参加了一项为期 8 天的开放性试验，接受三种剂量的 PB [250mg（第 I 组）、500mg（第 II 组）或 1000mg（第 III 组）]，每日两次（b.d.），同时给予维生素 D3{5000IU 每日一次（o.d.）}，PB（500mg b.d.）（第 IV 组）或维生素 D3（5000IU o.d.）（第 V 组），口服 4 天。于第 0 天、第 4 天和第 8 天采集血液；分离血浆，培养外周血单核细胞（PBMC）、非贴壁淋巴细胞（NAL）和 MDM。通过 qPCR 和 ELISA 分别测定细胞中的 LL-37 转录物和上清液中的肽浓度。在血浆中，通过 ELISA 测定 25- 羟基维生素 D3 水平。采用常规培养方法检测 MDM 对结核分枝杆菌（Mtb）（H37Rv）的杀伤作用。
第 II 组的 MDM 在第 4 天表现出增加的 LL-37 肽和转录物浓度，而第 I 组在第 4 天和第 8 天与第 0 天相比显示出增加的转录物（p<0.05）。与第 III 组和第 V 组相比，第 I 组和第 II 组在第 4 天的转录物水平更高（p<0.035）。与第 I 组和第 IV 组相比，第 II 组的淋巴细胞在第 4 天表现出更高水平的肽诱导（p<0.05），而第 IV 组在第 8 天的水平高于第 I 组和第 III 组（p<0.04）。与第 0 天相比，第 I 组、第 II 组和第 V 组在第 4 天对 Mtb 的细胞内杀伤明显增加（p<0.05）。
这些结果表明，每天两次给予 500mg PB 和每天一次给予 5000IU 维生素 D3 是诱导巨噬细胞和淋巴细胞中 LL-37 以及巨噬细胞内杀伤 Mtb 的最佳剂量。因此，该剂量具有治疗结核病的潜力，目前正在一项治疗活动性肺结核成人的临床试验中使用（NCT01580007）。
