Quan Zhou

Drug-induced liver injury in hospitalized patients with notably elevated alanine aminotransferase

AIM To identify the proportion, causes and the nature of drug-induced liver injury (DILI) in patients with notably elevated alanine aminotransferase (ALT). METHODS All the inpatients with ALT levels above 10 times upper limit of normal range (ULN) were retrospectively identified from a computerized clinical laboratory database at our hospital covering a 12-mo period. Relevant clinical information was obtained from medical records. Alternative causes of ALT elevations were examined for each patient, including biliary abnormality, viral hepatitis, hemodynamic injury, malignancy, DILI or undetermined and other causes. All suspected DILI cases were causality assessed using the Council for International Organizations of Medical Sciences scale, and only the cases classified as highly probable, probable, or possible were diagnosed as DILI. Comments related to the diagnosis of DILI in the medical record and in the discharge letter for each case were also examined to evaluate DILI detection by the treating doctors. RESULTS A total of 129 cases with ALT > 10 ULN were identified. Hemodynamic injury (n = 46, 35.7%), DILI (n = 25, 19.4%) and malignancy (n = 21, 16.3%) were the top three causes of liver injury. Peak ALT values were lower in DILI patients than in patients with hemodynamic injury (14.5 ± 5.6 ULN vs 32.5 ± 30.7 ULN, P = 0.001). Among DILI patients, one (4%) case was classified as definite, 19 (76%) cases were classified as probable and 5 (20%) as possible according to the CIOMS scale. A hepatocellular pattern was observed in 23 (92%) cases and mixed in 2 (8%). The extent of severity of liver injury was mild in 21 (84%) patients and moderate in 4 (16%). Before discharge, 10 (40%) patients were recovered and the other 15 (60%) were improved. The improved patients tended to have a higher peak ALT (808 ± 348 U/L vs 623 ± 118 U/L, P = 0.016) and shorter treatment duration before discharge (8 ± 6 d vs 28 ± 12 d, P = 0.008) compared with the recovered patients. Twenty-two drugs and 6 herbs were found associated with DILI. Antibacterials were the most common agents causing DILI in 8 (32%) cases, followed by glucocorticoids in 6 (24%) cases. Twenty-four (96%) cases received treatment of DILI with at least one adjunctive drug. Agents for treatment of DILI included anti-inflammatory drugs (e.g., glycyrrhizinate), antioxidants (e.g., glutathione, ademetionine 1,4-butanedisulfonate and tiopronin), polyene phosphatidyl choline and herbal extracts (e.g., protoporphyrin disodium and silymarin). Diagnosis of DILI was not mentioned in the discharge letter in 60% of the cases. Relative to prevalent cases and cases from wards of internal medicine, incident cases and cases from surgical wards had a higher risk of missed diagnosis in discharge letter [odds ratio (OR) 32.7, 95%CI (2.8-374.1), and OR 58.5, 95%CI (4.6-746.6), respectively]. CONCLUSION DILI is mostly caused by use of antibacterials and glucocorticoids, and constitutes about one fifth of hospitalized patients with ALT > 10 ULN. DILI is underdiagnosed frequently.

Vertical cross-sectional imaging of colonic dysplasia in vivo with multi-spectral dual axes confocal endomicroscopy.

Pathologists evaluate histology sectioned perpendicular to the tissue surface, or vertical cross-section. This orientation (XZ-plane) enables evaluation of mucosal differentiation in the basilar-to-luminal direction. Current endomicroscopes use a conventional (single axis) optical design.1 Imaging is limited to horizontal cross-sections (XY-plane) where the micro-anatomy is frequently similar across the field-of-view (FOV). In the dual axes configuration, light is delivered and collected off-axis, and images can be detected over a much larger range of intensities.2 Molecular images collected using fluorescence can improve specificity for disease detection and reveal functional properties about tissue.3 Proper interpretation of these images requires correlation with the micro-anatomy. We aim to demonstrate the simultaneous collection of two fluorescence images in vivo in vertical cross-sections using a dual axes confocal endomicroscope. An overlay of molecular and anatomical images from normal and dysplastic mouse colonic mucosa will be displayed in real time.

Design and Synthesis of Near-Infrared Peptide for in Vivo Molecular Imaging of HER2

We report the development, characterization, and validation of a peptide specific for the extracellular domain of HER2. This probe chemistry was developed for molecular imaging by using a structural model to select an optimal combination of amino acids that maximize the likelihood for unique hydrophobic and hydrophilic interactions with HER2 domain 3. The sequence KSPNPRF was identified and conjugated with either FITC or Cy5.5 via a GGGSK linker using Fmoc-mediated solid-phase synthesis to demonstrate flexibility for this chemical structure to be labeled with different fluorophores. A scrambled sequence was developed for control by altering the conformationally rigid spacer and moving both hydrophobic and hydrophilic amino acids on the C-terminus. We validated peptide specificity for HER2 in knockdown and competition experiments using human colorectal cancer cells in vitro, and measured a binding affinity of kd = 21 nM and time constant of k = 0.14 min(-1) (7.14 min). We used this peptide with either topical or intravenous administration in a preclinical model of colorectal cancer to demonstrate specific uptake in spontaneous adenomas and to show feasibility for real time in vivo imaging with near-infrared fluorescence. We used this peptide in immunofluorescence studies of human proximal colon specimens to evaluate specificity for sessile serrated and sporadic adenomas. Improved visualization can be used endoscopically to guide tissue biopsy and detect premalignant lesions that would otherwise be missed. Our peptide design for specificity to HER2 is promising for clinical translation in molecular imaging methods for early cancer detection.

Integrated monolithic 3D MEMS scanner for switchable real time vertical/horizontal cross-sectional imaging.

We present an integrated monolithic, electrostatic 3D MEMS scanner with a compact chip size of 3.2 × 2.9 mm(2). Use of parametric excitation near resonance frequencies produced large optical deflection angles up to ± 27° and ± 28.5° in the X- and Y-axes and displacements up to 510 μm in the Z-axis with low drive voltages at atmospheric pressure. When packaged in a dual axes confocal endomicroscope, horizontal and vertical cross-sectional images can be collected seamlessly in tissue with a large field-of-view of >1 × 1 mm(2) and 1 × 0.41 mm(2), respectively, at 5 frames/sec.

Multimodal laser-based angioscopy for structural, chemical and biological imaging of atherosclerosis

The complex nature of atherosclerosis demands high-resolution approaches to identify subtle thrombogenic lesions and define the risk of plaque rupture. Here, we report the proof-of-concept use of a multimodal scanning fiber endoscope (SFE) consisting of a single optical fiber scanned by a piezoelectric drive that illuminates tissue with red, blue, and green laser beams, and digitally reconstructs images at 30 Hz with high resolution and large fields-of-view. By combining laser-induced reflectance and fluorescence emission of intrinsic fluorescent constituents in arterial tissues, the SFE allowed us to co-generate endoscopic videos with a label-free biochemical map to derive a morphological and spectral classifier capable of discriminating early, intermediate, advanced, and complicated atherosclerotic plaques. We demonstrate the capability of scanning fiber angioscopy for the molecular imaging of vulnerable atherosclerosis by targeting proteolytic activity with a fluorescent probe activated by matrix metalloproteinases. We also show that the SFE generates high-quality spectral images in vivo in an animal model with medium-sized arteries. Multimodal laser-based angioscopy could become a platform for the diagnosis, prognosis, and image-guided therapy of atherosclerosis.

In vivo photoacoustic tomography of EGFR overexpressed in hepatocellular carcinoma mouse xenograft.

EGFR is a promising cell surface target for in vivo imaging that is highly overexpressed in hepatocellular carcinoma (HCC), a common cancer worldwide. Peptides penetrate easily into tumors for deep imaging, and clear rapidly from the circulation to minimize background. We aim to demonstrate use of an EGFR specific peptide to detect HCC xenograft tumors in mice with photoacoustic imaging. Nude mice implanted with human HCC cells that overexpress EGFR were injected intravenously with Cy5.5-labeled EGFR and scrambled control peptides respectively. Photoacoustic images collected from 0 to 24 h. Photoacoustic signal peaked in tumors at 3 h post-injection. Images from 0 to 1.8 cm beneath the skin revealed increased target-to-background (T/B) ratio from tumors. The T/B ratio was significantly greater for the EGFR versus control peptide. Clearance of signal was observed by ∼24 h. EGFR overexpression was validated with immunofluorescence and immunohistochemistry. A peptide specific for EGFR delivered systemically can detect HCC xenograft tumors in vivo with photoacoustic imaging.

Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope.

Confocal endomicroscopy is an emerging imaging technology that has recently been introduced into the clinic to instantaneously collect “optical biopsies” in vivo with histology-like quality. Here, we demonstrate a fast scanner located in the distal end of a side-viewing instrument using a compact lens assembly with numerical aperture of 0.5 to achieve a working distance of 100 μm and field-of-view of 300 × 400 μm2. The microelectromechanical systems (MEMS) mirror was designed based on the principle of parametric resonance and images at 5 frames per second. The instrument has a 4.2 mm outer diameter and 3 cm rigid length, and can pass through the biopsy channel of a medical endoscope. We achieved real time optical sections of NIR fluorescence with 0.87 μm lateral resolution, and were able to visualize in vivo binding of a Cy5.5-labeled peptide specific for EGFR to the cell surface of pre-cancerous colonocytes within the epithelium of dysplastic crypts in mouse colon. By performing targeted imaging with endomicroscopy, we can visualize molecular expression patterns in vivo that provide a biological basis for disease detection.

In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.

Targeted sections in either XY or XZ plane with dual-axes confocal endomicroscope (Conference Presentation)

We demonstrate a dual axes confocal architecture, which can be used to collect horizontal(XY-plane) or vertical cross-sectional(XZ-plane) images for tissue. This scanner head is 5.5mm in outer diameter(OD), and integrates a 3D MEMS scanner with a compact chip size of 3.2×2.9mm2. To realize the miniaturization, there are some obstacles of the small size of 3D MEMS scanner, MEMS wire bundle, the air pressure effect for MEMS motion, the processing of parabolic mirror, and optical alignment to come over. In our probe, separation mechanical structure for optical alignment was adopted and a step shape MEMS holder was designed to deal with the difficult of MEMS wire bundle. Peptides have been demonstrated tremendous potential for in vivo use to detect colonic dysplasia. This class of in vivo molecular probe can be labeled with near-infrared (NIR) dyes for visualizing the full depth of the epithelium in small animals. To confirm our probe performance, we take use of USAF 1951 resolution target to test its lateral and axial resolution. It has lateral and axial resolution of 2.49um and 4.98um, respectively. When we collect the fluorescence imaging of colon, it shows that the field of view are 1000um×1000um (horizontal) and 1000um×430um (vertical). The horizontal and vertical cross-sectional images of fresh mouse colonic mucosa demonstrate imaging performance with this miniature instrument.

Photoacoustic imaging of hepatocellular carcinoma targeting gold nanoshells (Conference Presentation)

Plasmonic gold nanoshell (GNS) probe penetrates into tumors for deep imaging, enables superior photoacoustic contrast. Glypican-3 (GPC3) specific peptide (Kd = 71 nM) conjugated gold nanoshell (λabs=770nm) was used to detect HCC xenograft tumors in mice with photoacoustic imaging. This targeting probe demonstrated tumor uptake after 1 hr and cleared in 12 hrs. Images at a mean (±SD) depth of 9.7±1.4 mm from 0 to 2.1 cm beneath the skin revealed increased PA signal from tumors. Highest tumor uptake and tumor to normal tissue ratio occurred at 2 hrs post injection (T/B = 4.45±0.22, n = 8). Molecular targeting GNS showed potential as a simple, effective and rapid technique for noninvasive in vivo monitoring HCC tumor growth and GPC3 expression.