Circulating cathepsin B and D in pregnancy.

The purpose of this prospective study was to investigate the changes in the circulating levels of cathepsin B and D in pregnancy. We obtained longitudinal cathepsin B and D levels in 76 healthy pregnant women in the first and third trimesters and compared these levels with 20 non-pregnant controls. The plasma levels of soluble cathepsin B and D were measured using an enzyme-linked immunosorbent assay kit. The cathepsin D concentrations in the third trimester were significantly higher than that in the first trimester (p < .001), and the cathepsin D levels in the first trimester were significantly lower than that in the non-pregnant controls (p = .002). However, there was no significant difference in the cathepsin B level throughout pregnancy compared to the non-pregnant controls. Our study is unique in evaluating the longitudinal changes in the cathepsin B and D levels in pregnancies without obstetric complications. The results implicate that changes in the levels of cathepsins might be essential in placentation. Therefore, molecular and genetic studies on cathepsin B and D are needed to understand the roles of these enzymes in pregnancy, thereby contributing to the understanding of placentation. Impact statement What is already known? Matrix metalloproteinases (MMP) have been widely studied, and their function is very important in the normal implantation process. The level of MMP-9 is known to increase throughout pregnancy, while the level of MMP-2 decreases in the first trimester. In addition to MMPs, other proteases are important for placental development; cathepsins B and D are two of the proteases that are involved in the normal placentation process. The function of cathepsin D is related to MMPs because this protease can activate MMPs either directly or indirectly. Nevertheless, the role of circulating cathepsins in pregnancy has not yet been fully elucidated. What do these results add? This study provides evidence, for the first time, that there are fluctuations of plasma cathepsin D level and there are no changes in the plasma cathepsin B level in a normal pregnancy. Moreover, we demonstrated that a cathepsin D level is significantly decreased in the first trimester compared to the non-pregnant controls, and that the level is markedly elevated in the third trimester. What are the implications of these findings for clinical practice and/or further research? Cathepsins B and D should be further studied locally in the placenta to explain the differences in the concentration of cathepsin D and no changes in cathepsin B, thereby exploring their exact roles.

The regulatory role of Dipeptidyl peptidase I on the activation of immune granulocytes.

Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, required for activation of serine proteases of granulocytes including mast cells (MCs), neutrophils (NPs) and others, which were found in synovial tissue of patients with rheumatoid arthritis (RA). But, the role of DPPI associated with those cells in RA development is unclear. In this study, the collagen-induced-arthritis (CIA) rat-model was employed to investigate the expression and activity levels of DPPI and its association with RA progress. Primary granulocytes were freshly extracted from bone-marrows of normal or CIA rats, human mast cell line LAD-2 and primary neutrophils, human-recombinant-DPPI, DPPI-inhibitor Gly-Phe-CHN2 , LTB4, anti-IgE antibody, calcium ionophore were used to study the regulatory role of DPPI in cell activations. The increased DPPI activities in synovial fluids, serum, and bone-marrow homogenates of CIA rats associated with RA severities progress were observed after injections. MMP2/9 expressions in SFs and bone-marrow were in different patterns. Regular-Blood-Tests have shown the high leveled DPPI activities associated with granulocytes differentiations in-vivo in blood of CIA rats. In-vitro cell models, DPPI up-regulated the proliferation of primary bone-marrow granulocytes of normal rats, but inhibited that of CIA rats. DPPI up-regulated and Gly-Phe-CHN2 down-regulated MCs intracellular DPPI and chymase activities. Gly-Phe-CHN2 also inhibited the LTB4 -activated-NPs and NP-elastase activities. Following stimulation of calcium ionophore, the net-releases of DPPI and ß-hexosaminidase from MCs were increased over a time-course, while Gly-Phe-CHN2 down-regulated MCs and NPs activation. Our findings demonstrate the role of DPPI in regulating MCs and NPs activation, and modulating proteolysis in the process of RA.

Serum based fluorescent assay for evaluating dipeptidyl peptidase I activity in collagen induced arthritis rat model.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease and derived from immune granule cells. It has been suggested playing an important role in the development of rheumatoid arthritis. In this study, a coumarin based fluorescent probe (GF-AFC) was designed and synthesized to evaluate DPPI activity in serum or tissue homogenates of collagen-induced arthritis (CIA) rats, an inflammatory arthropathy model. It was revealed that the fluorescent intensity was significantly increased in a very short time after specific substrate GF-AFC reacted with the DPPI. The fluorophore (AFC) was released to shine after the cleavage reaction which was examined by 19F NMR spectroscopy. It has been shown that DPPI hydrolyzed the GF-AFC in a robust, linear, and time dependent manner at a significant high rate. A serum-based DPPI activity assay was validated by spiking and gradient dilution methods, there were no interferences or auto-fluorescence observed. The Coefficient of Variance calculated for serum-based DPPI activity assays indicates the good reproducibility. The good correlation has been seen between serum DPPI levels and the severity of arthritis during RA development in CIA rats. Our study has demonstrated a new serum based diagnostic assay for detecting DPPI activity using coumarin conjugated fluorescent (GF-AFC) as a substrate. The successful implementation of the case would provide beneficial experience in rheumatoid arthritis research.

Evidence for a founder mutation in the cathepsin C gene in three families with Papillon-Lefèvre syndrome.

BACKGROUND: Papillon-Lefèvre syndrome (PLS; OMIM 245000) is a rare autosomal recessive disorder. Clinically, PLS is characterized by hyperkeratosis involving the palms, soles, elbows and knees which is followed later on by periodontitis, destruction of alveolar bone and loss of primary and permanent teeth. The condition is caused by mutations in the cathepsin C (CTSC) gene. METHODS: We analyzed the DNA of members from 3 consanguineous families for mutations in the CTSC gene by direct sequencing analysis. We then performed haplotype analysis. RESULTS: We identified an identical recurrent missense mutation, R272P, in all 3 families. Microsatellite marker analysis around the CTSC gene revealed the same haplotype on the mutation-carrying allele in all 3 families. CONCLUSION: The presence of this common mutation in families from 2 different geographical areas provides evidence for a founder effect for CTSC mutations in PLS.

Inhibition of dipeptidyl-peptidase IV does not increase circulating IGF-1 concentrations in growing pigs.

The enzyme dipeptidyl peptidase-IV (DPP-IV) inactivates a variety of bioactive peptides, including glucagon-like peptide-1 (GLP-1) and growth hormone releasing hormone (GHRH). Inhibiting DPP-IV in order to increase circulating GLP-1 is of interest as a treatment for Type II diabetes. Inactivation of DPP-IV may also increase circulating GHRH, potentially enhancing growth in domestic animals. To test the hypothesis that inhibition of DPP-IV activity will influence the growth hormone/ IGF-1 axis, growing pigs (Sus scrofa domesticus, 78 kg) were treated with a DPP-IV inhibitor (Compound 1, the 2,5-difluor-ophenyl analog of the triazolopiperazine MK0431, sitagliptin), and plasma concentrations of IGF-1 were monitored. Pigs were administered either sterile saline (0.11 ml/kg followed by a continuous infusion at 2 ml/hr for 72 hrs, controls, n = 2), Compound 1 (2.78 mg/kg followed by a continuous infusion at 0.327 mg/kg x hr for 72 hrs, n = 4) or GHRH (0.11 ml/kg sterile saline, followed by a continuous infusion of GHRH at 2.5 microg/ kg x hr for 48 hrs, n = 4). Plasma concentrations of Compound 1 were maintained at 1 microM, which resulted in a 90% inhibition of circulating DPP-IV activity. Relative to the predose 24-hr period, area under the IGF-1 concentration curve (AUC) tended to be lower (P = 0.062) with Compound 1 (.79 +/- 130 ng/ml x hr) than controls (543 +/- 330 ng/ml x hr). GHRH treatment increased the IGF-1 AUC (1210 +/- 160 ng/ml x hr, P = 0.049 vs. controls and P = 0.001 vs. Compound 1). We conclude that inhibition of DPP-IV does not alter the circulating levels of IGF-1 in the growing pig.

Papillon-Lefèvre syndrome: correlating the molecular, cellular, and clinical consequences of cathepsin C/dipeptidyl peptidase I deficiency in humans.

A variety of neutral serine proteases are important for the effector functions of immune cells. The neutrophil-derived serine proteases cathepsin G and neutrophil elastase are implicated in the host defense against invading bacterial and fungal pathogens. Likewise, the cytotoxic lymphocyte and NK cell granule-associated granzymes A and B are important for the elimination of virus-infected cells. The activation of many of these serine proteases depends on the N-terminal processing activity of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). Although mice deficient in DPPI have defects in serine protease activation in multiple cellular compartments, the role of DPPI for human serine protease activation is largely undefined. Papillon-Lefevre syndrome (PLS) is a rare autosomal recessive disease associated with loss-of-function mutations in the DPPI gene locus. In this study, we established that the loss of DPPI activity is associated with severe reduction in the activity and stability of neutrophil-derived serine proteases. Surprisingly, patients with PLS retain significant granzyme activities in a cytotoxic lymphocyte compartment (lymphokine-activated killer) and have normal lymphokine-activated killer-mediated cytotoxicity against K562 cells. Neutrophils from patients with PLS do not uniformly have a defect in their ability to kill Staphylococcus aureus and Escherichia coli, suggesting that serine proteases do not represent the major mechanism used by human neutrophils for killing common bacteria. Therefore, this study defines the consequences of DPPI deficiency for the activation of several immune cell serine proteases in humans, and provides a molecular explanation for the lack of a generalized T cell immunodeficiency phenotype in patients with PLS.

Innate immune response mechanisms in non-insulin dependent diabetes mellitus patients assessed by flow cytoenzymology.

It is well known that infections in patients with diabetes mellitus are more severe, although there is controversy for increased susceptibility to them. Non-specific immune response mechanisms could be related to defense and/or susceptibility to pathogens. The aim of this study was to investigate the activity of several enzymes involved in the primary host defense mechanisms in non-insulin dependent diabetes mellitus (NIDDM). Twenty NIDDM females with a mean HbA(1c) level of 8.19% were included. No patient had clinical evidence of infection. As controls 20 healthy females were studied. The enzymes tested were dipeptidyl-peptidase I (DPP-I), cathepsin B and D, NADPH oxidase and superoxide dismutase (oxidative burst) and collagenase. Isolated leukocytes were incubated with the specific substrates in pyrogen free conditions. The intracellular enzyme activity was analyzed by flow cytometry. Collagenase enzymatic activity was similar in the three leukocyte subpopulations studied. Oxidative burst induction in monocytes was comparable between both groups. Enzyme activity of cathepsin B and D in all cell subsets, oxidative burst in PMN cells, and DPP-I in lymphocytes and monocytes from patients, was higher than those from healthy females (P<0.05). Overall, our findings demonstrate an enhanced functional status of several intracellular leukocyte enzymes in NIDDM. Furthermore, the increased oxidative burst induction and the consequent production of free radicals, may contribute to vascular complications. Other mechanisms - either from the non-specific or specific immune response - deserve investigation to establish if diabetic patients are more susceptible to infectious diseases.

Identification of cathepsin C mutations in ethnically diverse papillon-Lefèvre syndrome patients.

INTRODUCTION: Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder characterised by palmoplantar keratoderma and severe, early onset periodontitis, which results from deficiency of cathepsin C activity secondary to mutations in the cathepsin C gene. To date, 13 different cathepsin C mutations have been reported in PLS patients, all of which are homozygous for a given mutation, reflecting consanguinity. AIM: To evaluate the generality of cathepsin C mutations in PLS, we studied an ethnically diverse group of 20 unrelated families. METHODS: Mutations were identified by direct automated sequencing of genomic DNA amplified for exonic regions and associated splice site junctions of the cathepsin C gene. Long range PCR was performed to determine the genomic structure of the cathepsin C gene. RESULTS: The cathepsin C gene spans over 46 kb, with six introns ranging in size from 1.6 to 22.4 kb. Eleven novel mutations and four previously reported mutations were identified in affected subjects from 14 families. Missense mutations were most common (9/15), followed by nonsense mutations (3/15), insertions (2/15), and deletions (1/15). Among these 14 probands, two were compound heterozygotes. Affected subjects with transgressions of the dermal lesions onto the knees or elbows or both had mutations in both the pro- and mature regions of the enzyme, although most were in the mature region. CONCLUSION: Mutations in the mature region of cathepsin C were more likely to be associated with the transgressions of the dermatological lesions, although the results were not statistically significant. A comprehensive list of all cathepsin C mutations described to date, representing 25 mutations from 32 families with PLS and related conditions, is also presented.