Alexander Goihl

Role of dipeptidyl peptidase IV (DP IV)-like enzymes in T lymphocyte activation: investigations in DP IV/CD26-knockout mice.

Abstract Background: Dipeptidyl peptidase IV (DP IV, CD26) and DP IV-like enzymes, such as dipeptidyl peptidase II (DP II), dipeptidyl peptidase 8 (DP8), and dipeptidyl peptidase 9 (DP9), have been recognized to regulate T lymphocyte activation. Lys[Z(NO2)]-thiazolidide (LZNT) and Lys[Z(NO2)]-pyrrolidide (LZNP), non-selective inhibitors of DP IV-like activity known to target DP IV as well as DP II, DP8, and DP9, suppress T lymphocyte proliferation in vitro. Moreover, these inhibitors are capable of attenuating the severity of autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and experimental arthritis, a model of human rheumatoid arthritis, in vivo, particularly in combination with inhibitors of aminopeptidase N (APN, CD13) enzymatic activity. Methods: Here, we studied the influence of non-selective and selective inhibitors of DP IV-like enzymes on DNA synthesis in mitogen-stimulated splenocytes from wild-type C57BL/6 mice and DP IV/CD26-knockout (DP IV/CD26-KO) mice. Results: LZNT and LZNP, the non-selective inhibitors of DP IV-like activity, suppressed the DNA synthesis in stimulated splenocytes from wild-type and DP IV/CD26-KO mice to a comparable extent. Further, a selective inhibitor of DP8/DP9 activity was capable of suppressing DNA synthesis in mitogen-stimulated splenocytes of both wild-type and knockout mice to the same extent. In contrast, selective inhibitors of DP IV and DP II lacked this suppressive activity. Conclusions: Our data support the hypothesis that DP8 and/or DP9 represent additional pharmacological targets for the suppression of T cell proliferation and for anti-inflammatory therapy. Clin Chem Lab Med 2009;47:268–74.

Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease

BACKGROUND Crohns disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB). METHODS Human GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD). RESULTS Anti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p<0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p<0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmens rho=0.493, p<0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p=0.0307). CONCLUSIONS Anti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD.

Insulin and insulin-like growth factor-1 can modulate the phosphoinositide-3-kinase/Akt/FoxO1 pathway in SZ95 sebocytes in vitro

A recent hypothesis suggests that a high glycaemic load diet-associated increase of insulin-like growth factor-1 (IGF-1) and insulin may promote acne by reducing nuclear localization of the forkhead box-O1 (FoxO1) transcription factor via activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. Using SZ95 sebocytes as a model, we investigated the effect of the most important insulinotropic western dietary factors, IGF-1 and insulin on acne. SZ95 sebocytes were stimulated with different concentrations of IGF-1 and insulin (0.001, 0.01, 0.1 and 1 μM) for 15 to 120 min ± PI3K inhibitor LY294002 (50 μM). Cytoplasmic and nuclear protein expression of p-Akt and p-FoxO1 as well as FoxO transcriptional activity was analysed. In addition, the proliferation and differentiation of sebocytes and their TLR2/4 expression were determined. We found that high concentrations of IGF-1 and insulin differentially stimulate the PI3K/Akt/FoxO1 pathway by an early up-regulation of cytoplasmic p-Akt and delayed up-regulation of p-FoxO1 resulting in FoxO1 shift to the cytoplasm and the reduction of FoxO transcriptional activity, physiological serum concentration had no effect. IGF-1 at concentrations of 0.1 and 1 μM significantly reduced proliferation but increased differentiation of sebocytes to a greater extent than insulin (0.1 and 1 μM), but up-regulated TLR2/4 expression to comparable extent. These data provide the first in vitro evidence that FoxO1 principally might be involved in the regulation of growth-factor-stimulatory effects on sebaceous lipogenesis and inflammation in the pathological condition of acne. However, the in vivo significance under physiological conditions remains to be elucidated.

Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia

BackgroundCerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia.MethodsMale Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test.ResultsQualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged.ConclusionsDistinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets.

The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I).

The pancreatic zymogen granule membrane protein (GP2) is expressed by pancreatic acinar cells and M cells of the ileum. GP2 is the closest related homologue of the urine resident Tamm–Horsfall protein (THP). Recently, it was shown that THP is a ligand of various scavenger receptors (SRs). Therefore, we were interested, if GP2 has similar properties. cDNA of different SRs was stably transfected into a murine thymoma cell line. GP2 was recombinantly expressed, purified and biotinylated. Binding or uptake of GP2 by transfected cells or monocyte-derived dendritic cells (moDCs) was analyzed by flow-cytometry. GP2 is a binding partner of the scavenger receptor expressed on endothelial cells I (SREC-I) but not of SR-AI and SR-BI. The dissociation constant (Kd) of GP2 binding to SREC-I is 41.3 nM. SREC transfected cells are able to internalize GP2. moDCs express SREC-I and also bind and internalize GP2. Inhibition of SREC-I on moDCs with anti-SREC-I antibodies does not result in a decreased GP2 binding. Interaction of GP2 with SREC-I and uptake might have profound effects in antigen clearance and mediation of the immune response. In addition to SREC-I other presently unknown receptors for GP2 on DCs might be involved in this process.

Dual Inhibition of Dipeptidyl Peptidase IV and Aminopeptidase N Suppresses Inflammatory Immune Responses

Abstract:  The ectopeptidases dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) are known to regulate T cell activation. Since selective inhibitors of DP IV and APN suppress DNA synthesis and cytokine production of stimulated T cells in a TGF‐β1‐dependent manner, we tested whether combined application of DP IV and APN inhibitors enhances this immunomodulatory effect. The results show that simultaneous application of DP IV and APN inhibitors significantly suppressed DNA synthesis in mitogen‐ or anti‐CD3‐stimulated human T cells in vitro when compared to the use of a single DP IV or APN inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF‐β1 production associated with the observed immunosuppressive effects. In vivo, targeting both DP IV and APN led to a potent treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). This review summarizes the evidence for the role of both enzymes in T cell activation in vitro and in vivo and provides a rationale for using combined and dual peptidase inhibitors to treat autoimmune diseases like MS.

Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence

Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double‐strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose‐ and time‐dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose‐dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage.

Evidence of Crohn’s disease-related anti-glycoprotein 2 antibodies in patients with celiac disease

Abstract Background: Autoantibodies to exocrine-pancreatic glycoprotein 2 (anti-GP2) are Crohn’s disease (CD) markers. However, CD-specific antibodies have also been found in celiac-disease (CeD) patients, in which type 1 diabetes-specific autoantibodies against endocrine pancreatic targets can be present. We investigated whether anti-GP2 are also present in CeD, a disease like CD which is also characterised by intestinal mucosal inflammation with barrier impairment. Methods: Antibodies against GP2, tissue transglutaminase (tTG), deamidated gliadin (dGD), glutamic decarboxylase (GAD), and islet antigen-2 (IA2) were tested in sera from 73 CD patients, 90 blood donors (BD), and 79 (58 de novo) CeD patients (2 consecutive sera were available from 40 patients). Results: IgA and/or IgG anti-GP2 were found in 15/79 (19.0%) CeD patients on at least one occasion, in 25/73 (34.2%) CD patients, and in 4/90 (4.4%) BD (CeD vs. CD, p=0.042; BD vs. CeD and CD, p<0.001, respectively). Amongst the 58 de novo CeD patients, anti-GP2 IgA and/or IgG were present in 11 (19.0%). Anti-GP2 IgA was significantly less prevalent in CeD compared with CD (p=0.004). Anti-GP2 IgA and IgG in CD patients demonstrated a significantly higher median level compared to patients with CeD (p<0.001, p=0.008, respectively). IgA anti-GP2 levels correlated significantly with IgA anti-tTG and anti-dGD levels in CeD Spearman’s coefficient of rank correlation (ρ)=0.42, confidence interval (CI): 0.26–0.56, p<0.001; ρ=0.54, CI 0.39–0.65, p<0.001, respectively. Conclusions: The presence of anti-GP2 in CeD patients supports the notion that loss of tolerance to GP2 can probably be a manifestation of an autoinflammatory process in this intestinal disorder.

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice.

Abstract Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, ‘peptidase-targeted Immunoregulation’ (PETIR™), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR™ in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR™ represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.

Methodologic issues in the measurement of interleukin-16 in clinical blood samples using immunoassays.

Quantitation of interleukin-16 (IL-16) in clinical blood samples has strongly increased, since IL-16 appears to be involved in the pathogenesis of several inflammatory diseases. IL-16 is synthesized in the cell cytoplasm as precursor protein (pro-IL-16), which can be processed by caspase-3 into N-terminal (N-IL-16) and C-terminal (C-IL-16) fragments. C-IL-16 is described to be subsequently secreted. Using commercially available IL-16 ELISA, a pro-IL-16 ELISA and immunoprecipitation analysis, we investigated, whether type and handling of blood samples influence IL-16 quantitation and whether existing IL-16 ELISA are specific for C-IL-16. We observed that cell-rich plasma samples reflect falsely-elevated IL-16 concentrations due to cell contaminations. Interestingly, not C-IL-16, but pro-IL-16 represents the major IL-16 form in cell-rich plasma samples. Notably, commercially IL-16 ELISA could not distinguish between C-IL-16 and pro-IL-16. Thus, cell-rich plasma samples should not be used for IL-16 measurements and new methods are necessary for quantitation of C-IL-16 and pro-IL-16 uniquely.