Bao Tung Pham

Evaluation of fibrosis in precision-cut tissue slices

1. In this review, the use of precision-cut tissue slices (PCTS) of the liver, kidney, lung and intestine in fibrosis research are evaluated and future possibilities are discussed. 2. In vivo models or techniques that are applicabless to be investigated in PCTS are discussed. 3. It is concluded that the early onset of fibrosis can be induced successfully in PCTS prepared from human and experimental animals. 4. Moreover, precision-cut slices of fibrotic tissue are effective in gaining new knowledge of the mechanisms of fibrosis and of the mode of action of potential antifibrotic drugs. 5. Both healthy and fibrotic human tissue slices will pave the way for the testing of novel therapeutic drugs to treat patients with fibrosis avoiding interspecies extrapolation.

Precision‐cut rat, mouse, and human intestinal slices as novel models for the early‐onset of intestinal fibrosis

Intestinal fibrosis (IF) is a major complication of inflammatory bowel disease. IF research is limited by the lack of relevant in vitro and in vivo models. We evaluated precision‐cut intestinal slices (PCIS) prepared from human, rat, and mouse intestine as ex vivo models mimicking the early‐onset of (human) IF. Precision‐cut intestinal slices prepared from human (h), rat (r), and mouse (m) jejunum, were incubated up to 72 h, the viability of PCIS was assessed by ATP content and morphology, and the gene expression of several fibrosis markers was determined. The viability of rPCIS decreased after 24 h of incubation, whereas mPCIS and hPCIS were viable up to 72 h of culturing. Furthermore, during this period, gene expression of heat shock protein 47 and plasminogen activator inhibitor 1 increased in all PCIS in addition to augmented expression of synaptophysin in hPCIS, fibronectin (Fn2) and TGF‐β1 in rPCIS, and Fn2 and connective tissue growth factor (Ctgf) in mPCIS. Addition of TGF‐β1 to rPCIS or mPCIS induced the gene expression of the fibrosis markers Pro‐collagen1a1, Fn2, and Ctgf in both species. However, none of the fibrosis markers was further elevated in hPCIS. We successfully developed a novel ex vivo model that can mimic the early‐onset of fibrosis in the intestine using human, rat, and mouse PCIS. Furthermore, in rat and mouse PCIS, TGF‐β1 was able to even further increase the gene expression of fibrosis markers. This indicates that PCIS can be used as a model for the early‐onset of IF.

Precision-cut kidney slices (PCKS) to study development of renal fibrosis and efficacy of drug targeting ex vivo

ABSTRACT Renal fibrosis is a serious clinical problem resulting in the greatest need for renal replacement therapy. No adequate preventive or curative therapy is available that could be clinically used to target renal fibrosis specifically. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, we here evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study the development of fibrosis and its prevention using anti-fibrotic compounds. Precision-cut slices (200-300 μm thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml) for 48 h in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate targeted to PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-SMA, fibronectin, collagen I and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 h of incubation, and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression, which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. A major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue. Drug Discovery Collection: TGFβ induces renal fibrosis in ex vivo cultured precision-cut kidney slices, which can be attenuated by IFNγ.

Organ- and species-specific biological activity of rosmarinic acid

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis.

P024 Regional differences in effect of TGF-beta1 and PDGF on the early onset of intestinal fibrosis

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300-400 μm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.

P067 Human and rodent ex vivo model for intestinal fibrosis in inflammatory bowel disease (IBD)

Background: One of the major complications in IBD is intestinal fibrosis (IF). It is the result of the chronic inflammation of intestinal tissue. IF causes narrowing of the intestinal lumen and potential stricture formation. For the study of the cellular and molecular mechanism of IF in IBD adequate animal models are lacking. Our aim is to develop an ex vivo model for IF by using human and rodent precision-cut intestinal slice (PCIS). In PCIS all cell types are present in their original tissue-matrix environment and can be used as a model to study the early onset of IF. Methods: Rat, mouse and human jejunum were excised and prepared as a segment embedded in agarose. PCIS (estimated 300-400 mm) was prepared and incubated up to 24 hr (rat) or 48 hr (human and mouse). ATP content of the PCIS was used to assess the general viability. Moreover, morphology (rat and human) was evaluated. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), Heat Shock Protein 47 (HSP47), alpha-Smooth Muscle Actin (SMA), connective tissue growth factor (CTGF), Synaptophysin (SYN) and Fibronectin (FIB) were determined. Results: Mouse PCIS were viable up to 48 hr. However, for rat and human PCIS ATP content was decreased to 25% (24hr) and 70% (48 hr), respectively. ATP content of rat and human PCIS correlated well with morphology of the PCIS. In rat PCIS, after 24 hr, HSP47 (3.2 fold) and FIB (2.1 fold) gene expressions were significantly increased. In the presence of 5 ng/mL TGF-β, COL1A1 (1.8 fold), SMA (1.5 fold) and CTGF (2.1 fold) were significantly up-regulated compared to 24 hr control. Meanwhile HSP47 gene expression was slightly decreased (0.8 fold). Similarly, in mouse PCIS, gene expression of HSP47 (3.9 fold) and FIB (4.3 fold) was significantly increased after 48 hr. When incubated with 5 ng/mL TGF-β, COl1A1 and FIB were significantly up-regulated (2.0 fold) compared to 48 hr control, HSP47 and CTGF gene expression were slightly, but significantly increased (1.2 fold). In human PCIS, after 48 hr, HSP47 (3.5-fold) and SYN (2.5 fold) were significantly up-regulated. However, incubation of human PCIS with 5 ng/ml of TGF-β, none of the investigated fibrosis genes was affected. Conclusions: In rat, mouse and human PCIS an increase in gene expression of early-onset of fibrosis makers was found. In addition, TGF-β was able to induce fibrosis markers in rat and mouse, but not in human PCIS. Rodent and human PCIS are promising ex vivo models to study the early onset of intestinal fibrosis.

Quantitative Comparison of the Neutralizing Capacity of Therapeutic Anti-TNF-α Drugs and the Cross-Reactivity of Anti-Anti-TNF-α Antibodies

Background and Aims: Identification of non-adherence is an important component of shortand long-term management of IBD. A validated screening tool for non-adherence in IBD would help determine those patients who may be at highest risk for non-adherent behavior and potentially worse disease outcomes. Our aim was to determine if scores from a selfreported adherence survey of IBD patients correlated with pharmacy refill data, as a reliable measure of medication adherence. Methods: The self-reported tool was an 8-item survey, the Moritsky Medication Adherence Scale, which has been previously validated in patients with hypertension. Each question is worth a point with a maximum score of 8. A score of <4 was defined as a patient at high risk for non-adherence, and a score of 7-8 at no risk for non-adherence. The study was IRB-approved and informed consent was obtained. Surveys were completed at the time of a routine clinic appointment. Patient pharmacies were contacted at 3 time points: time of enrollment for refill information regarding the previous 3 months, then 3 months and 6 months from enrollment. Medications discontinued due to intolerance or non-response before the 6-month time period were excluded. Refill data were recorded for each time interval as the Medication Possession Ratio (MPR) and adherence was defined as greater than 80%. Analysis of variance with Pearsons correlation coefficient was used to determine the relationship between survey scores and MPR by drug class. Results: One hundred fifty consecutive patients were enrolled. Ninety-four patients had Crohns disease and 56 had ulcerative colitis. Eighty-nine patients (59%) were female. Thirty six percent of patients were on a 5-ASA, 41% on a thiopurine, 11% on infliximab, 6% on an injectable biologic, and 5% on budesonide. The median adherence score was 7 (range, 0-8). Fiftytwo percent stated they rarely missed a dose of medication. However, the adherence by refill data ranged from 25% to 71% by drug class. Only those on a thiopurine had a survey score that positively correlated with adherence (p = 0.02). For all other medication classes, there was no correlation between reported scores and refill data either prior to or 6 months following survey completion. Conclusion: Overall, adherence with IBD therapies was low. Only those on a thiopurine were likely to have an adherence score that predicted high refill behavior. Adherence with therapy for IBD is complex and cannot be predicted in a reliable manner by a self-reported survey tool validated for other chronic conditions.