Tenofovir disoproxil fumarate (tenofovir) has been associated with renal dysfunction in people infected with human immunodeficiency virus (HIV) receiving combination antiretroviral therapy. We reviewed data from an HIV preexposure prophylaxis trial to determine if tenofovir use was associated with changes in renal function in an HIV-uninfected population.
Host-microbial interactions are of major importance in the pathogenesis of inflammatory bowel disease (IBD). Toll-like receptors (TLR) are pattern recognition receptors which recognise conserved molecular patterns derived from micro-organisms. Crypt intestinal epithelial cells (IEC) were isolated form mucosal specimens of healthy controls and patients with IBD (ulcerative colitis, UC, and Crohn's disease). A population of IEC enriched for intestinal stem cells (ISC) were identified using Hoechst dye exclusion and by their adherence to cultured primary intestinal myofibroblast cell monolayers.
Compared to healthy control colon, TLR2 and TLR4 mRNA and surface protein were significantly up-regulated in crypt IEC isolated from the inflamed mucosa of UC and Crohn's colitis. Compared to healthy control ileum, TLR4 mRNA was significantly up-regulated in crypt epithelial cells isolated from the inflamed mucosa of Crohn‟s ileitis. TLR2 and TLR4 mRNA expression from histologically normal and inflamed colonic mucosa in UC did not significantly differ, and expression of TLR4 transcripts was significantly greater in crypt IEC isolated from histologically normal proximal colonic mucosal samples compared to healthy controls. Myofibroblast-adherent crypt cells expressed TLR2 and TLR4 protein to a greater level than the underlying myofibroblasts. Hoechst-effluxing putative intestinal stem cells expressed both TLR2 and TLR4 transcripts and protein, and TLR3 and TLR5 transcripts.
In conclusion, crypt intestinal epithelial cells up-regulated TLR2 and TLR4 expression in UC and Crohn's colitis and up-regulated TLR4 expression in Crohn's ileitis. TLR2 and TLR4 was expressed constitutively in crypt IEC from histologically normal mucosa, suggesting differential TLR expression may in part be a primary event in UC. This provides further insights into the pathogenesis of IBD. Putative intestinal stem cells expressed TLR2, TLR3, TLR4 and TLR5, suggesting that direct microbial sensing by ISC may be important in maintaining intestinal homeostasis and in regulating ISC function.
TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine. We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn9s disease (CD) crypt intestinal epithelial cells (IEC).
Methods
Ileal and colonic mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (CD or UC). Additionally, paired samples from histologically normal (proximal colon) and inflamed (distal colon) mucosa were obtained from five colectomy specimens with isolated left-sided UC. Crypt IECs were isolated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. Cell surface TLR protein was studied by flow cytometry (expressed as median fluorescent intensity, MFI). TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (IQR).
Results
Relative to normal controls, TLR2 mRNA expression in IEC was significantly elevated in inflamed colonic UC [3.18-fold (1.03–10.40), p=0.003] and inflamed colonic CD [3.45 (0.80–5.40), p=0.012]. TLR4 mRNA was significantly elevated in normal colonic UC [1.90 (1.69–4.31], p=0.017), inflamed colonic UC [2.33 (1.15–4.45), p=0.024], inflamed colonic CD [1.71 (1.00–4.32), p=0.042] and inflamed ileal CD [1.84 (1.43–4.66), p=0.03]. In paired analyses, IEC TLR2 and TLR4 mRNA expression was not significantly different in normal UC mucosa compared to inflamed UC mucosa. Compared to IEC from normal control colon, IEC from colonic CD showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4–29.0) vs 33.1 (17.7–107.5), p=0.05] and TLR4 protein [MFI 12.1 (4.9–25.3) vs 40.9 (23.2–103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt IEC.
Conclusion
IEC expression of TLR4 mRNA was up-regulated in inflamed and un-inflamed colonic and ileal IBD. TLR2 mRNA was up-regulated in inflamed colonic IBD. Interestingly, TLR2 and TLR4 mRNA expression was similar in IECs isolated from un-inflamed, histologically normal mucosa as it was in IECs isolated from inflamed mucosa in UC. This may suggest TLR2 and TLR4 up-regulation is a primary rather than secondary event in UC. Moreover, TLR2 and TLR4 protein was expressed in greater amounts on the surface of IECs in CD.
Abstract The macrophage contents of 40 human primary breast cancers were ascertained using three separate macrophage membrane markers. Four prognostic factors (lymph node status, tumour size, histological grade and oestrogen receptor status) were determined concurrently in these mammary carcinomas. No significant relationship was found between the macrophage content of the mammary carcinomas and any individual prognostic factor. When the patients were grouped according to whether they had 0–2 or 3–4 high risk factors, however, the macrophage content was significantly greater in those tumours with the poorer prognosis, regardless of the marker used to enumerate the cells. A high macrophage content, therefore, is not associated with favourable prognostic factors in breast cancer. Indeed, the present study has revealed the reverse to be the case.
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