SummaryThe cathepsins D (CTSD), B (CTSB) and L (CTSL) are important for the intracellular degradation of proteins. Increased cathepsin expression is associated with inflammatory diseases. We have shown previously an induction of CTSD expression in intestinal macrophages (IMAC) in inflamed mucosa of patients with inflammatory bowel disease (IBD). Here we investigated the regulation of CTSB and CTSL in IMAC during IBD and effects of CTSD and CTSB/CTSL inhibition in vivo. Human IMAC were isolated from normal and inflamed mucosa. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for CTSB and CTSL mRNA. Immunostaining was used to confirm PCR results. Cathepsin inhibition was investigated in the dextran-sulphate-sodium (DSS) colitis model in mice with application of pepstatin A (CTSD inhibitor), CA-074 (CTSB inhibitor) and Z-Phe-Tyraldehyde (CTSL inhibitor). CTSL mRNA was significantly up-regulated in IMAC isolated from IBD mucosa. Up-regulated protein expression was found mainly in areas of mucosal damage by immunostaining. Inhibition of CTSD in mouse DSS colitis was followed by an amelioration of the disease. Inhibitor-treated mice showed a significant lower histological score (HS) and less colon reduction in comparison to controls. Similarly, simultaneous inhibition of CTSB/CTSL was followed by a significant amelioration of colitis. Expression of tissue-degrading cathepsins is increased in IMAC in IBD. Inhibition of CTSD as well as CTSB/CTSL is followed by an amelioration of experimental colitis. The prevention of mucosal damage by cathepsin inhibition could represent a new approach for the therapy of IBD.

SUMMARYDown-regulation of receptors involved in the recognition or transmission of inflammatory signals and a reduced responsiveness support the concept that macrophages are 'desensitized' during their differentiation in the intestinal mucosa. During inflammatory bowel disease (IBD) intestinal macrophages (IMACs) change to a reactive or 'aggressive' type. After having established a method of isolation and purification of IMACs, message for cathepsin D was one of the mRNAs we found to be up-regulated in a subtractive hybridization of Crohn's disease (CD) macrophages versus IMACs from control mucosa. The expression of cathepsin D in intestinal mucosa was analysed by immunohistochemistry in biopsies from IBD and control patients and in a mouse model of dextran sulphate sodium (DSS)-induced acute and chronic colitis. IMACs were isolated and purified from normal and inflamed mucosa by immunomagnetic beads armed with a CD33 antibody. RT-PCR was performed for cathepsin D mRNA. Results were confirmed by Northern blot and flow cytometrical analysis. Immunohistochemistry revealed a significant increase in the cathepsin D protein expression in inflamed intestinal mucosa from IBD patients compared to non-inflamed mucosa. No cathepsin D polymerase chain reaction (PCR) product could be obtained with mRNA from CD33-positive IMACs from normal mucosa. Reverse transcription (RT)-PCR showed an induction of mRNA for cathepsin D in purified IMACs from IBD patients. Northern blot and flow cytometry analysis confirmed these results. Cathepsin D protein was also found in intestinal mucosa in acute and chronic DSS-colitis but was absent in normal mucosa. This study shows that expression of cathepsin D is induced in inflammation-associated IMACs. The presence of cathepsin D might contribute to the mucosal damage in IBD.

Summary Bispecific antibodies offer the possibility of improving effector‐cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single‐chain Fv antibody (bsscFv), directed against FcγRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single‐chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti‐CD16 and the anti‐HLA class II scFv components of the bsscFv were 8·6 × 10−8 mol/l and 13·7 × 10−8 mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody‐dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv‐mediated specific lysis of both HLA class II‐positive, malignant human B‐lymphoid cell lines and primary cells from patients with chronic B‐cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv‐antibody is an efficient molecule for effector‐cell mediated lysis of malignant human B‐lymphoid cells.

Background: The glycoprotein (gp) 96 links the adaptive with the innate immune system. It is a chaperone with a binding domain for peptides generated by proteasomal degradation. During cellular stress, peptide loaded gp96 can be released and presented to T cells by antigen presenting cells (APCs). Methods: mRNAs from in vitro differentiated macrophages (iv mac) and normal intestinal macrophages (IMACs) were compared by subtractive hybridisation and Affymetrix GeneChip analysis. Differentiation induced expression of gp96 was investigated in the multicellular spheroid (MCS) model. In vivo gp96 protein expression was detected by double labelling immunohistochemistry of human colon and in the CD4 + CD62L + T cell transfer mouse model. Results: Five of 76 clones obtained by subtractive hybridisation revealed .99% sequence homology to gp96. Affymetrix GeneChip analysis confirmed induction of gp96 in IMACs. Gp96 mRNA was detected in IMACs from normal and intestinal bowel disease mucosa. Induction of gp96 protein was observed after seven days in the MCS model of IMAC differentiation. Immunohistochemistry confirmed the presence of gp96 protein in IMACs in normal mucosa as well as in mucosa from patients with ulcerative colitis and diverticulitis. In mucosa from Crohn's disease (CD) patients, gp96 protein was not detectable. In the CD4

Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3α, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3α protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3α. FACS of purified IMAC clearly indicated expression of MIP-3α in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3α protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3α during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3α is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3α expression could be involved in the recruitment of CD45R0+ cells into the lamina propria.

Background: Biomarkers for the prognosis in severe illness are commonly used. Mean platelet volume (MPV) is an easily accessible haematological parameter which may reflect platelet activation as well as endothelial damage. There are only a few reports concerning the use of this parameter as a biomarker in severe illness.Purpose of the study was the evaluation of MPV as a prognostic biomarker in patients with sepsis.Methods: Patients with suspected sepsis attending the Infectious Diseases wards, medical ICU or interdisciplinary anesthesiology ICU were prospectively enrolled in the analysis. Measures included demographics, medical history as well as clinical and laboratory biomarker determinations on admission and/or first symprtomatic day and day of positive blood culture. Patients were followed having outcomes and biomarkers assessed in the course.Results: 183 patients (66 female) with confirmed sepsis were available for the analysis. Median age was 71 years (range 21-97 years), median BMI was 26.3 kg/m 2 . All had at least one premorbid medical conditions, more than two third had >3 significant pre-existing diseases. Bacteria most commonly detected were S. aureus (54/183) and E. coli (39/183) reflecting the origin of sepsis which mostly were device-related and endocarditis (in 37/183), urogenital (in 35/183) or skin, soft-tissues and bone/joints (27/183), respectively. Unfavourable outcomes (death) at day 30 had 27/183 (14.8%). Patients who died had a higher MPV at admission compared to the survivors (9.6 vs. 9.19 fl, p = 0.031) which increased to an MPV of 11.2 vs. 9.7 fl (p = 0.008) at the time of positive blood culture drawn. Only mean arterial pressure (MAP) at the detection of bacteremia (p = 0.02) and thrombocyte count at bacteremia (p = 0.033) had significant associations with death. Neither temperature, leukocytes, lactate, PCT or CrP had significant associations (all p > 0.1). Receiver operator curve showed AUC for MPV at admission of 0.71 and MPV at bacteremia of 0.68. MAP and thrombocytes at bacteremia had much lower AUC (0.39 and 0.36, respectively).Conclusion: These preliminary data confirm the potential usefulness of MPV as an easily accessible prognostic marker in sepsis. Further studies are required to confirm our results.

These findings indicate an essential role of gp96 in the maintenance of tolerance against luminal antigens in the intestinal mucosa. The absence of gp96 in intestinal macrophages of patients with Crohn's disease might provoke loss of this tolerance mediating mechanism.

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