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We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.

Osteolytic bone disease in multiple myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising response rates in relapsed and newly diagnosed MM, and bortezomib has recently been reported to inhibit OCLs. We here investigated the effect of lenalidomide on OCL formation and osteoclastogenesis in comparison with bortezomib. Both drugs decreased αVβ3-integrin, tartrate-resistant acid phosphatase-positive cells and bone resorption on dentin disks. In addition, both agents decreased receptor activator of nuclear factor-κB ligand (RANKL) secretion of bone marrow stromal cells (BMSCs) derived from MM patients. We identified PU.1 and pERK as major targets of lenalidomide, and nuclear factor of activated T cells of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was observed. We demonstrated a significant decrease of growth and survival factors including macrophage inflammatory protein-α, B-cell activating factor and a proliferation-inducing ligand. Importantly, in serum from MM patients treated with lenalidomide, the essential bone-remodeling factor RANKL, as well as the RANKL/OPG ratio, were significantly reduced, whereas osteoprotegerin (OPG) was increased. We conclude that both agents specifically target key factors in osteoclastogenesis, and could directly affect the MM-OCL-BMSCs activation loop in osteolytic bone disease.

Background Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) is a treatment option for peritoneal surface malignancies. The ability to detect microscopic foci of peritoneal metastasis intraoperatively may ensure the completeness of cytoreduction. In this study, we evaluated the suitability of a hand-held cathepsin-based fluorescent imaging system for intraoperative detection of appendiceal and colorectal peritoneal metastasis. Methods Peritoneal tumors and normal peritoneal tissues were collected from patients with appendiceal and colorectal peritoneal metastasis. Expression of different cathepsins (CTS-B, -D, -F, -G, -K, -L, -O, and -S) was determined by quantitative RT-PCR and immunohistochemistry. The hand-held cathepsin-based fluorescent imaging system was used to detect peritoneal xenografts derived from human colon cancer cells (HT29, LoVo and HCT116) in nu/nu mice. Results While the expression levels of CTS-B, -D, -L, and -S could be higher in peritoneal tumors than normal peritoneum with a median (range) of 6.1 (2.9–25.8), 2.0 (1.0–15.8), 1.4 (0.8–7.0), and 2.1 (1.6–13.9) folds by quantitative RT-PCR, respectively, CTS-B was consistently the major contributor of the overall cathepsin expression in appendiceal and colonic peritoneal tumors, including adenocarcinomas and low-grade appendiceal mucinous neoplasms. Using peritoneal xenograft mouse models, small barely visible colonic peritoneal tumors (<2.5 mm in maximum diameter) could be detected by the hand-held cathepsin-based fluorescent imaging system. Conclusions Because cathepsin expression is higher in peritoneal tumors than underlying peritoneum, the hand-held cathepsin-based fluorescent imaging system could be useful for intraoperative detection of microscopic peritoneal metastasis during CRS-HIPEC and clinical trial is warranted.

Background. In cystic fibrosis (CF) the upper (UAW) and lower airways (LAW) are reservoirs for pathogens like Pseudomonas aeruginosa. The consecutive hosts’ release of proteolytic enzymes contributes to inflammation and progressive pulmonary destruction. Objectives were to assess dynamics of protease : antiprotease ratios and pathogens in CF-UAW and LAW sampled by nasal lavage (NL) and sputum before and after intravenous- (IV-) antibiotic therapy. Methods. From 19 IV-antibiotic courses of 17 CF patients NL (10 mL/nostril) and sputum were collected before and after treatment. Microbiological colonization and concentrations of NE/SLPI/CTSS (ELISA) and MMP-9/TIMP-1 (multiplex bead array) were determined. Additionally, changes of sinonasal symptoms were assessed (SNOT-20). Results. IV-antibiotic treatment had more pronounced effects on inflammatory markers in LAW, whereas trends to decrease were also found in UAW. Ratios of MMP-9/TIMP-1 were higher in sputum, and ratios of NE/SLPI were higher in NL. Remarkably, NE/SLPI ratio was 10-fold higher in NL compared to healthy controls. SNOT-20 scores decreased significantly during therapy ( P = 0.001 ) . Conclusion. For the first time, changes in microbiological patterns in UAW and LAW after IV-antibiotic treatments were assessed, together with changes of protease/antiprotease imbalances. Delayed responses of proteases and antiproteases to IV-antibiotic therapy were found in UAW compared to LAW.

Background: AA amyloidosis develops in patients with chronic inflammatory diseases. The AA amyloid proteins are proteolytic fragments obtained from serum amyloid A (SAA). Previous studies have provided evidence that endosomes or lysosomes might be involved in the processing of SAA, and contribute to the pathology of AA amyloidosis. Objective: To investigate the anatomical distribution of cathepsin (Cath) B and CathL in AA amyloidosis and their ability to process SAA and AA amyloid proteins. Methods and results: CathB and CathL were found immunohistochemically in every patient with AA amyloidosis and displayed a spatial relationship with amyloid in all the cases studied. Both degraded SAA and AA amyloid proteins in vitro. With the help of mass spectrometry 27 fragments were identified after incubation of SAA with CathB, nine of which resembled AA amyloid proteins, and seven fragments after incubation with CathL. CathL did not generate AA amyloid-like peptides. When native human AA amyloid proteins were used as a substrate 26 fragments were identified after incubation with CathB and 18 after incubation with CathL. Conclusion: The two most abundant and ubiquitously expressed lysosomal proteases can cleave SAA and AA amyloid proteins. CathB generates nine AA amyloid-like proteins by its carboxypeptidase activity, whereas CathL may prevent the formation of AA amyloid proteins by endoproteolytic activity within the N-terminal region of SAA. This is particularly interesting, because AA amyloidosis is a systemic disease affecting many organs and tissue types, almost all of which express CathB and CathL.

AimsCathepsin V (CTSV/CTSL2) is a lysosomal cysteine proteinase and plays a role in extracellular matrix degradation. It is associated with poor prognosis in invasive breast cancer (IBC), but its role in breast ductal carcinoma in situ (DCIS) remains unclear. In this study, we aimed to evaluate the prognostic significance of CTSV in DCIS.MethodsCTSV protein expression was immunohistochemically assessed in a well-characterised and annotated cohort of DCIS comprising pure DCIS (n=776) and DCIS coexisting with IBC (n=239). CTSV expression was analysed in tumour cells and surrounding stroma, including its association with clinicopathological parameters and outcome.ResultsIn pure DCIS, high CTSV expression was observed in 29% of epithelial tumour cells and 20% of surrounding stroma. High expression in both components was associated with features of poor prognosis including higher nuclear grade, hormone receptor negativity and HER2 positivity. In addition, stromal CTSV expression was associated with larger DCIS size, comedo-type necrosis and high proliferation index. DCIS associated with IBC showed higher CTSV expression than pure DCIS either within the epithelial tumour cells or surrounding stroma (p<0.0001 and p=0.001, respectively). In DCIS/IBC, CTSV expression was higher in the invasive component than DCIS component either in tumour cells or surrounding stroma (both p<0.0001). CTSV stromal expression was associated with invasive recurrence independent of other prognostic factors in patients treated with breast conserving surgery (HR=3.0, p=0.005).ConclusionHigh expression of CTSV is associated with poor outcome in DCIS and is a potential marker to predict DCIS progression to invasive disease.

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo .

Increased expression of cathepsins has diagnostic as well as prognostic value in several types of cancer. Here, we demonstrate a novel magnetic resonance imaging (MRI) method, which uses poly-L-glutamate (PLG) as an MRI probe to map cathepsin expression in vivo, in a rat brain tumor model. This noninvasive, high-resolution and non-radioactive method exploits the differences in the CEST signals of PLG in the native form and cathepsin mediated cleaved form. The method was validated in phantoms with known physiological concentrations, in tumor cells and in an animal model of brain tumor along with immunohistochemical analysis. Potential applications in tumor diagnosis and evaluation of therapeutic response are outlined.

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.