Cysteine-type cathepsins promote the effector phase of acute cutaneous delayed-type hypersensitivity reactions.

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

Reproducibility and Comparability of Preclinical PET Imaging Data: A Multicenter Small-Animal PET Study.

The standardization of preclinical imaging is a key factor to ensure the reliability, reproducibility, validity, and translatability of preclinical data. Preclinical standardization has been slowly progressing in recent years and has mainly been performed within a single institution, whereas little has been done in regards to multicenter standardization between facilities. This study aimed to investigate the comparability among preclinical imaging facilities in terms of PET data acquisition and analysis. In the first step, basic PET scans were obtained in 4 different preclinical imaging facilities to compare their standard imaging protocol for 18F-FDG. In the second step, the influence of the personnel performing the experiments and the experimental equipment used in the experiment were compared. In the third step, the influence of the image analysis on the reproducibility and comparability of the acquired data was determined. Distinct differences in the uptake behavior of the 4 standard imaging protocols were determined for the investigated organs (brain, left ventricle, liver, and muscle) due to different animal handling procedures before and during the scans (e.g., fasting vs. nonfasting, glucose levels, temperature regulation vs. constant temperature warming). Significant differences in the uptake behavior in the brain were detected when the same imaging protocol was used but executed by different personnel and using different experimental animal handling equipment. An influence of the person analyzing the data was detected for most of the organs, when the volumes of interest were manually drawn by the investigators. Coregistration of the PET to an MR image and drawing the volume of interest based on anatomic information yielded reproducible results among investigators. It has been demonstrated that there is a huge demand for standardization among multiple institutions.

The administration route of tumor-antigen-specific T-helper cells differentially modulates the tumor microenvironment and senescence.

Cancer treatment with adoptively transferred tumor-associated antigen-specific CD4+ T-helper cells is a promising immunotherapeutic approach. In the pancreatic cancer model RIP-Tag2, the intraperitoneal (i.p.) application of Tag-specific TH1 cells exhibited a profound antitumoral efficiency. We investigated, whether an intravenous (i.v.) application of Tag-TH1 cells induces an equivalent therapeutic effect. Adoptively transferred fluorescent Tag-TH1 cells revealed a pronounced homing to the tumors after either i.p. or i.v. transfer, and both routes induced an almost equivalent therapeutic effect as demonstrated by magnetic resonance imaging, blood glucose level course and histology. The i.v. administration of Tag-TH1 cells induced p16INK4-positive/Ki67-negative tumor senescence more efficiently than i.p. administration. Both routes replenish host CD4+ T cells by transferred T cells and recruitment of B and dendritic cells to the tumors while reducing CD8+ T cells and depleting macrophages. Both administration routes efficiently induced a similar antitumoral efficiency despite the pronounced senescence induction after i.v. administration. Thus, a combinatory i.v./i.p. injection of therapeutic cells might overcome limitations of the individual routes and improve therapeutic efficacy in solid tumors.