Dipeptidyl Peptidase IV

Dipeptidyl-peptidase IV/CD26 (DPIV) is a 110 kD cell-surface protease belonging to the prolyloligopep­tidase family. It selectively removes the N-terminal dipeptide from peptides with proline or alanine in the second position. Apart from its catalytic activity, it interacts with several proteins, for instance, adenosine deaminase, the HIV gp120 protein, fibronectin, collagen, the chemokine receptor CXCR4, and the tyrosine phosphatase CD45. DPIV is expressed on a specific set of T lymphocytes, where it is up-regulated after activation. It is also expressed in a variety of tissues, primarily on endothelial and epithelial cells. A soluble form is present in plasma and other body fluids.

DPIV has been proposed as a diagnostic or prognostic marker for various tumors, hematological malignancies, immunological, inflammatory, psychoneuroen­docrine disorders, and viral infections.

DPIV truncates many bioactive peptides of medical importance. It plays a role in glucose homeostasis through proteolytic inactivation of the incretins. DPIV inhibitors improve glucose tolerance and pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients. DPIV has become a novel target for Type II diabetes therapy.

The role of DPIV CD26 within the immune system is a combination of its exopeptidase activity and its interactions with different molecules. This enables DPIV CD26 to serve as a co-stimulatory molecule to influence T cell activity and to modulate chemotaxis. DPIV is also implicated in HIV-1 entry, malignant transformation, and tumor invasion.

DPIV is the archetypal member of its six-member gene family. Four members of this family, DPIV, FAP (fibroblast activation protein), DP8 and DP9, have a rare substrate specificity, hydrolysis of a prolyl bond two residues from the N-terminus.

The crystal structure shows that the soluble form of DPIV comprises two domains, an alpha/beta-hydrolase domain and an eight-blade beta-propeller domain. The propeller domain contains the ADA (adenosine deaminase) binding site, a dimerization site, antibody epitopes and two openings for substrate access to the internal active site. DPIV is active as a homodimer. Glycosylation of DPIV is not a prerequisite for catalysis, dimerization, or ADA binding.

DPIV has a variety of peptide substrates, the best studied being GLP-1 (glucagon-like peptide-1), NPY (neuropeptide Y), CXCL12, RANTES, stromal cell-derived factor-1 and eotaxin.

It could be shown that inhibition of the catalytic activity can provoke many cellular effects, including induction of tyrosine phosphorylations and p38 MAP kinase activation as well as suppression of DNA synthesis and reduced production of various cytokines. TGF-beta 1, the production and secretion of which is increased after DP IV inhibition, supposedly mediates the observed suppressive effects by maintaining p27kip expression levels which leads to a cell cycle arrest in G1. Moreover, anti-CD3-induced signalling pathways, including Ca2+ mobilisation, MEK1-, Erk1/2– and PKB-activation, can be strongly affected by DPIV inhibition. Thus, the enzymatic activity or at least the interaction of effectors with the catalytic domain of CD26 seems to be important for crucial functions of this cell surface antigen.

Dipeptidyl Peptidase IV regulation

In cultured human dermal fibroblasts, DPIV was consistently upregulated by IL-1alpha and IL-1beta, but consistently downregulated by glucocorticoids, tumor necrosis factor alpha (TNFalpha) and transforming growth factor beta(1) (TGFbeta(1)).

In the immature murine T-cell line, R1.1, dynorphin-A(1–17) down-regulated DPIV in a dose-dependent manner.

Interleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes.

In a study, 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPIV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity.

High glucose increases expression and activity of DPIV, possibly contributing to GLP-1 reduction in type 2 diabetic patients.

In a study, changes of DPIV in the context of leptin or leptin receptor deficiency were assessed . C57BL/6 mice, Leptin-deficient mice (ob/ob mice, B6.V-Lep<ob>) and Leptin-receptor-deficient mice (db/db mice, B6.Cgm+/+ Lepr) were infected with B. Calmette-Guerin (BCG) and sacrificed three days later. DPP IV activity in serum was higher in ob/ob mice and in db/db mice than in wild-type mice. The expression of DPIV/CD26 on splenocytes was higher in ob/ob mice than in wild-type animals, and lower in db/db mice, and decreased upon stimulation with BCG in ob/ob mice only. Several T cell antigens including CTLA-4 were expressed aberrantly in ob/ob and in db/db mice. These observations provide evidence for a relationship between DPIV and leptin.

Dipeptidyl Peptidase IV in Diabetes and Obesity

Mice lacking the gene encoding DPIV (DPIV-/-) are refractory to the development of obesity and hyperinsulinemia. Pair-feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to high fat diet-induced obesity in the DPIV- /- mice. Ablation of DPIV also is associated with elevated GLP-1 levels and improved metabolic control in these animals, resulting in improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin- induced loss of beta cell mass and hyperglycemia. Together, these observations suggest that chronic deletion of DPIV gene has significant impact on body weight control and energy homeostasis, providing validation of DPIV inhibition as a viable therapeutic option for the treatment of metabolic disorders related to diabetes and obesity.

Dipeptidyl Peptidase IV Inhibitors

These drugs reversibly block DPIV-mediated inactivation of incretin hormones, for example, glucagon-like peptide 1 (GLP-1) and also other peptides that have alanine or proline as the penultimate N-terminal amino acid. DPIV inhibitors, therefore, increase circulating levels and prolong the biological activity of endogenous GLP-1, but whether this is sufficient to fully explain the substantial reduction in haemoglobin A(1c) (HbA(1c)) and associated metabolic profile remains open to further investigation. DPIV inhibitors such as vildagliptin and sitagliptin have been shown to be highly effective antihyperglycaemic agents that augment insulin secretion and reduce glucagon secretion via glucose-dependent mechanisms. There seems to be no change in body weight, and very low rates of hypoglycaemia and gastrointestinal disturbance with these agents.

DPIV inhibitors represent a major new class of oral antidiabetic drug and their metabolic profile offers a number of unique clinical advantages for the management of type 2 diabetes.

Dipeptidyl Peptidase IV and Immunity

DPIV/CD26 role in immune regulation has been extensively characterized, with recent findings elucidating its linkage with signaling pathways and structures involved in T-lymphocyte activation as well as antigen presenting cell-T-cell interaction.

DPIV has been implicated in the regulation of T cell activation and function. Th1 cells express three- to sixfold more CD26 protein than Th2 cells. However, DPIV activity was found similar in both cell populations at physiological substrate concentrations because of differences in K(m) and V(max) values of DPIV on Th1 and Th2 cells.

sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPIV enzyme activity. It was also shown that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. These findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.

Recombinant soluble DPIV/CD26 enhances peripheral blood T-cell proliferation induced by the recall antigen tetanus toxoid (TT).DPIV/CD26 binds caveolin-1 on antigen-presenting cells (APC), and residues 201–211 of DPIV/CD26 along with the serine catalytic site at residue 630, which constitute a pocket structure of DPIV/CD26, contribute to binding to caveolin-1 scaffolding domain. In addition, following DPIV/CD26-caveolin-1 interaction on TT-loaded monocytes, caveolin-1 is phosphorylated, with linkage to NF-kappaB activation, followed by upregulation of CD86. Finally, reduced caveolin-1 expression on APC inhibits DPIV/CD26-mediated CD86 upregulation and abrogates DPIV/CD26 effect on TT-induced T-cell proliferation, and immunohistochemical studies revealed an infiltration of DPIV/CD26+ T cells in the sub-lining region of rheumatoid synovium and high expression of caveolin-1 in the increased vasculature and synoviocytes of the rheumatoid synovium. Taken together, these results strongly suggest that DPIV/CD26– cavolin-1 interaction plays a role in the upregulation of CD86 on TT-loaded APC and subsequent engagement with CD28 on T cells, leading to antigenspecific T-cell activation such as the T-cell-mediated antigen-specific response in rheumatoid arthritis.

DPIV/CD26 truncates several chemokines as well as neuropeptides and influences immune responses via modulation of cell adhesion and T cell activation, suggesting an involvement of DPIV/CD26 inflammation.

The truncated RANTES(3–68) lacked the ability of native RANTES(1–68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colonystimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3–68) showed a reduced activity, relative to that of RANTES(1–68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. These results indicate that DPIV/CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, Th2 lymphocytes, and basophils. Human eotaxin has a penultimate proline, indicating that it might be a substrate for dipeptidyl-peptidase IV. Eotaxin is efficiently cleaved by DPIV/CD26 and the NH2-terminal truncation affects its biological activity. DPIV/CD26-truncated eotaxin(3–74) showed reduced chemotactic activity for eosinophils and impaired binding and signaling properties through the CC chemokine receptor 3. Moreover, eotaxin(3–74) desensitized calcium signaling and inhibited chemotaxis toward intact eotaxin. This physiological processing may be an important down-regulatory mechanism, limiting eotaxin-mediated inflammatory responses.

A remarkably reduced T cell recruitment was seen in a model of asthma using rat strains that either lack or exhibit reduced DPIV/CD26-like enzymatic activity, suggesting a role for CD26 in the pathogenesis of asthma via T celldependent processes such as antibody production.

It was shown by flow cytometry and by enzymatic DPIV assay that myelin basic protein (MBP)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis (MS) express high levels of DP IV/CD26. The enzymatic activity of resting TCC was found to be three to fourfold higher than on resting peripheral blood T cells and close to that of T cells 48 hours after phytohemaglutinin (PHA) stimulation. The DPIV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide suppress in a dose-dependent manner DNA synthesis and IFN-gamma, IL-4, and TNF-alpha production of the antigen- stimulated TCC. These data suggest that DPIV/CD26 plays a role in regulating activation of autoreactive TCC. Further in vivo investigations will clarify, whether the inhibition of the enzymatic activity of DPIV could be a useful tool for therapeutic interventions in MS and/or other autoimmune diseases.

In vivo administration of the reversible DPIV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) decreased and delayed clinical and neuropathological signs of adoptive transfer experimental autoimmune encephalomyelitis (EAE). I40 blocked DPIV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DPIV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.

In a rat model, acute rejection was associated with increased serum DPIV activity (p < 0.005). Specific inhibition abrogated acute (p < 0.0001) and accelerated (p < 0.01) rejection, impairing cytotoxicity and allospecific Igsynthesis. DPIV/CD26 is pivotal in T-cell mediated immune responses toward allo-Ag.

Experimental nephritis was induced by anti-Thy-1.1 monoclonal antibody E30 using Wistar or DPIV-lacking F344 rats. The application of a new monoclonal antibody against DPIV, F16, completely suppressed E30-induced proteinuria and mesangial proliferation in Wistar rats, whereas these preventive effects of F16 were not observed in F344 rats, which spontaneously lack DPIV protein. Treatment with F16 inhibited glomerular deposition of complement C3 and complement C4 after the binding of E30 to the mesangial cell surface.

Because the preventive effect of F16 was attributable to suppression of the complement cascade, its influences on complement-dependent mesangial cell lysis in vitro was examined. It was discovered that the complement cascade was markedly inactivated in F16-treated Wistar rat serum but not in F16-treated F344 rats. These results indicate that DPIV may play a somewhat crucial role in regulating the complement cascade and that inhibition of DPIV may serve as a new target for preventing complement-dependent tissue injury.

Dipeptidyl Peptidase IV and Cancer

Recent work also suggests that DPIV/CD26 has a significant role in tumor biology, being both a marker of disease behavior clinically as well as playing an important role in tumor pathogenesis and development.

DPIV/CD26 is aberrantly expressed in many cancers and plays a key role in tumorigenesis and metastasis. Its diverse cellular roles include modulation of chemokine activity by cleaving dipeptides from the chemokine NH(2)-terminus, perturbation of extracellular nucleoside metabolism by binding the ectoenzyme adenosine deaminase, and interaction with the extracellular matrix by binding proteins such as collagen and fibronectin.

Plasminogen type 2 (Pg 2) receptor in highly invasive 1-LN human prostate tumour cell line is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV. Pg 2 has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2gamma, Pg 2delta and Pg 2epsilon glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca(2+) signalling cascade

Prostate

DPIV loss is associated with increased bFGF production in metastatic prostate cancer cells. DPIV reexpression in prostate cancer cells blocks nuclear localization of bFGF, reduces bFGF levels, inhibits mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)1/2 activation, and decreases levels of urokinase-type plasminogen activator, known downstream effectors of bFGF signaling pathway. These molecular changes were accompanied by induction of apoptosis, cell cycle arrest, inhibition of in vitro cell migration, and invasion. Silencing of DPIV by small interfering RNA resulted in increased bFGF levels and restoration of mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)1/2 activation. These results indicate that DPIV inhibits the malignant phenotype of prostate cancer cells by blocking bFGF signaling pathway.

Non Small Cell Lung Cancer

Both mRNA and protein DPIV levels in non small cell lung cancer (NSCLC) cell lines and normal human bronchial epithelial cells were studied. DPIV expression was detectable in normal lung epithelial cells, but was absent or markedly reduced in all NSCLC cell lines at both mRNA and protein levels. Restoration of DPIV expression in NSCLC cells resulted in profound morphologic changes, inhibition of cell proliferation, anchorage-independent growth, in vitro cell migration and tumorigenicity in nude mice. DPIV reexpression also correlated with increased p21 expression, leading to induction of apoptosis and cell cycle arrest in G1 stage. These effects were accompanied by increased expression of cell surface proteins, fibroblast-activating protein (Fapalpha) and CD44 that are associated with suppression of tumor growth and metastasis. Thus, DPIV functions as a tumor suppressor, and its downregulation may contribute to the loss of growth control in NSCLC cells.

F344JAP rats lack the dipeptidyl peptidase IV activity of CD26 and show a reduced cell surface expression of the mutated CD26 glycoprotein. In vivo adhesion of vital dye-labeled MADB106 tumor cells, tumor colonization, CD26 enzymatic activity, and CD26 immunoreactivity in lungs and soluble CD26– like protein expression in serum were markedly reduced in F344JAP rats. These findings demonstrate that DPIV/CD26 protein expression exerts a key role in lung metastasis. In addition, NK cell cytotoxicity against MADB106 cells was diminished in the mutant F344 substrain, suggesting that DPIV/CD26 enzymatic activity sustains NK cytotoxicity. Interestingly, tumor cells lacked DPIV/CD26 immunoreactivity in vitro, but displayed DPIV/CD26 immunoreactivity in situ after in vivo inoculation as well as after incubation with rat serum, indicating that soluble CD26-like protein assembles in tumor cells during in vivo passage, which may interact with the process of tumor adhesion and metastasis. Overall, these findings indicate that altered expression and function of a single enzyme-the DPIV/CD26 protein can drastically change the outcome of metastatic disease.

Ovary

Ovarian carcinoma cell lines with higher DPIV expression were found to be less invasive. Furthermore, DPIV overexpression in SKOV3 cells, derived from serous cystadenocarcinoma, with little DPIV expression induced a dramatic change in cellular morphology and a significant decrease in the abilities of both migration and invasion. In addition, it was also shown that nude mice inoculated with DPIV-transfected SKOV3 cells showed significantly less peritoneal dissemination and longer survival time than those receiving the parental or vector-only transfected cells (mean survival time, 64.9 +/- 4.7, 35.7 +/- 2.8, and 36.6 +/- 1.8 days, respectively). This evidence implies that DPIV may functionally suppress peritoneal dissemination in ovarian carcinoma.

The introduction of DPIV cDNA into SKOV3 enhanced the expression of Ecadherin and beta-catenin, with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype. In addition, matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase, important markers associated with invasive and metastatic potential, were remarkably reduced. In contrast, tissue inhibitors of matrix metalloproteinases were up-regulated by DPIV transfection. Furthermore, suppression of the phosphorylation levels of mitogen-activated protein kinase isoform, extracellular signal-regulated kinase, was observed in DPIV-overexpressing cells. Thus, increasing DPIV expression may contribute to prolonged survival by upregulation of E-cadherin and tissue inhibitors of matrix metalloproteinases.

A study showed the possible correlation between DPIV/CD26 expression and less migratory potential with decreased MMP-2 expression in ovarian carcinoma cells. Moreover, induction of DPIV/CD26 resulted in reduced expressions of MMP-2 and mesenchymal markers such as vimentin and SMA, with a less invasive potential and an epithelial morphologic change. This evidence implies that DPIV/CD26 may play a crucial role in the antimetastatic potential in ovarian carcinoma.

Breast

A novel adhesion receptor/ligand pair was shown to mediate lung vascular arrest and metastasis of rat breast cancer cells. The interacting adhesion molecules are endothelial dipeptidyl peptidase IV and tumor cell surface-associated, polymeric fibronectin (FN). A truncated DPIV (DPIV(31–767): amino acids 31–767) in which the FN-binding site is preserved was shown to mask the breast cancer cell surface-associated FN complexes, causing a dosedependent inhibition of adhesion to endothelial DPIV and impeding lung colony formation by approximately 80%. Since surface accumulation of FN is chiefly occurring during dissemination in the blood and since many cancer cell types have surface receptors by which they may initiate FN accumulation on their surfaces, the present anti-metastatic treatment modality may extend its efficacy farther than appreciated by this study.

Mesothelium

DPIV enzyme activity in mesothelial cells was enhanced by the addition of ascites obtained from ovarian carcinoma patients in a time- and concentration- dependent manner, and flow cytometry and immunocytochemistry also revealed an increased expression of DPIV on the cell surface of mesothelial cells. The <3-kD fraction of malignant ascites increased the DPIV enzyme activity to the same level as the total ascites. Northern hybridization demonstrated that DPIV mRNA was increased 3-fold by the addition of the <3-kD malignant ascites. In conclusion, DPIV is highly expressed in human mesothelial cells and was regulated by ascites.

Melanoma

Tetracycline-inducible expression vectors were constructed to express DPIV in human melanoma cells. Reexpressing DPIV in melanoma cells at or below levels expressed by normal melanocytes induced a profound change in phenotype that was characteristic of normal melanocytes. DPIV expression led to a loss of tumorigenicity, anchorage-independent growth, a reversal in a block in differentiation, and an acquired dependence on exogenous growth factors for cell survival.

DPIV down-regulation is found in transformed melanocytes where nearly 100% of melanomas lack DPIV expression. Expression of a proteolytically active form of the DPIV protein inhibits the invasiveness of malignant melanoma cell lines lacking endogenous DPIV expression.

Glia

DPIV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III) and U87 glioblastoma multiforme (Grade IV) lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPIV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further studies of function of this enzyme in human glioma cells.

Endometrium

Immunohistochemical analyses showed that DPIV was strongly or moderately stained in glandular cells of the normal secretory phase. In endometrial adenocarcinoma, the DPIV expression decreased with advancing grade (P <.01).

Thyroid

There is no expression of DPP4 in normal healthy thyroid, while it is highly expressed in papillary thyroid carcinoma (PTC), as shown by gene expression profiling.

Dipeptidyl Peptidase IV and Mental Disorders

DPIV-deficient rats exhibited increased pain sensitivity in a non-habituated hot plate test, indicative of a reduced stress-induced analgesia. In line with this finding, reduced stress-like responses in tasks like the open field (OF), social interaction (SI), and passive avoidance test were found. Differences in DPIV-like activity appear to be involved in neurophysiological processes because DPIV-deficient animals were less susceptible to the sedative effects of ethanol. The varying phenotypes of the F344 substrains are likely to be mediated by differential degradation of DPIV substrates such as substance P, glucagon-like peptide (GLP)-1, enterostatin, and especially neuropeptide Y (NPY). Potentially, DPIV-deficient substrains represent an important tool for biomedical research, focusing on the involvement of DPIV and its substrates in behavioral and physiological processes.

Anorexia and Bulimia nervosa

DPIV activity was measured in the serum of 21 women with anorexia nervosa, 21 women with bulimia nervosa and 18 normal women. Serum DPIV activity was significantly lower in patients with anorexia nervosa and bulimia nervosa than in the normal controls. In the total study group, there were significant and inverse relationships between serum DPIV activity and the total scores on the Bulimic Investigatory Test, Edinburgh, the Eating Disorder Inventory (EDI) and the Hamilton Depression Rating Scale. In the total study group, no significant correlations between DPIV and age, body weight or body mass index could be found. It is concluded that lowered serum DPIV activity takes part in the pathophysiology of anorexia and bulimia nervosa. It is hypothesised that a combined dysregulation of DPIV and neuroactive peptides, which are substrates of DPIV, e.g. neuropeptide Y and peptide YY, could be an integral component of eating disorders.

In another study, serum DPIV activity and the number of CD26 (DPP IV)-positive peripheral blood lymphocytes were measured in 44 patients (anorexia nervosa (AN): n = 21, bulimia (B): n = 23) in four consecutive weekly analyses. The analysis of CD26-positive cells included the characterization of CD26-bright and CD26-dim positive subsets. Additionally, the expression of CD25 (IL-2 Receptor alpha chain) was evaluated to estimate the degree of T cell activation. The same analyses were carried out in healthy female volunteers (HC, n = 20). CD26-positive cells were reduced in patients as compared to healthy controls (mean 40.2% (AN) and 41.1% (B) vs. 47.4% (HC), p < 0.01), while the DPIV activity in serum was elevated (mean 108.4 U/l (AN) and 91.1 U/l (B) vs. 80.3 U/l (HC), p < 0.01).

Phobic Anxiety

The association between DPIV/sCD26 levels and phobic anxiety was tested using simple correlation analysis, linear regression and multivariate logistic regression analysis. A highly significant inverse association was found between DPIV/sCD26 concentrations and anxiety scores both in simple correlation and linear regression analysis. Compared with subjects in the first tertile of DPIV/sCD26 levels, the age-adjusted odds ratio for scoring >/=6 compared to scoring 0 or 1 was 0.31 (95% CI: 0.18–0.74) for the second and 0.47 (95% CI: 0.34–0.63) for the third tertile. Altogether, these data suggest that reduced plasma DPIV/sCD26 concentrations could be a marker of high levels of phobic anxiety in women.

Reactive Depression

DPIV activities were measured in patients with hepatitis C before and 2, 4 and 16 weeks after starting IFN alpha-based immunotherapy. Patients with lower baseline PEP or DPIV had significantly higher MADRS (Montgomery Asberg Depression Rating Scale) and HAM-A (Hamilton Anxiety Rating Scale) scores both at baseline and during immunotherapy. Patients with lower baseline DPIV had significantly higher increases in the MADRS following IFN alpha treatment. Thus, lower baseline PEP and DPP IV predict higher depressive and anxiety ratings during IFN alpha-based immunotherapy.

Dipeptidyl Peptidase IV and Natriuretic peptides

Human BNP (1–32), A-type natriuretic peptide 1–28 (ANP 1–28), and related peptides were incubated with purified DPIV and with human plasma. Cleavage products were analyzed by mass spectrometry. BNP (1–32) was cleaved by purified DPIV with a specificity constant of 0.37×10(6) L.mol(-1).s(-1). The DPIV activity in EDTA-plasma was able to truncate BNP (1–32) ex vivo. Addition of Vildagliptin, a specific DPIV inhibitor, prevented this truncation in a concentration-dependent manner.

Dipeptidyl Peptidase IV and Placenta

DPIV is expressed on human placental cytotrophoblasts. DPIV is important for the noninvasive extra-villous trophoblast (EVT) phenotype and the downregulation of this enzyme was strongly associated with migration or invasive EVT phenotype.

Preeclamptic placentas with intrauterine growth restriction showed significantly higher levels of activity than the controls (p < 0.05). It was proposed that placental DPIV influences fetal metabolism via the degradation of fetoplacental circulating bioactive peptides, including incretins, resulting in the regulation of fetal growth.

The role of DPIV activity and CD26-positive decidual lymphocytes in murine stress-triggered abortions by inhibition of DPIV activity was investigated. DBA/2-mated CBA mice were stressed on day 5.5 of pregnancy and received daily injections of an inhibitor of DPIV activity, Ile-thiazolidide (20 micromol/ kg). On day 13 of gestation, the animals were sacrificed and the percentage of implants and abortions documented. CD26-positive lymphocytes in spleen and uterine decidua and the intracellular cytokines interferon (IFN)- gamma and interleukin (IL)-10 were determined by flow cytometry. Stressed and nonstressed animals receiving an inactive stereoisomeric form were used as controls. In mice receiving the DPIV inhibitor, stress failed to boost the abortion rate (37.2% versus 13.6%, P < 0.01). IFN-gamma producing cells were increased in stressed animals, but returned to the baseline upon the inhibition of DPIV. The number of IL-10 producing cells was reduced in stressed animals, independent from DPIV inhibition.

These data indicate that DPIV may play a critical role in pregnancy failure by inducing a Th1 local response. The possible participation of DPIV in the onset of human spontaneous abortion (SA) was investigated. The systemic (peripheral blood) and local (decidua) percentages of CD4(+), CD8(+), CD26(+) and CD56(+) cells as well as the number of Th1 lymphocytes (CCR5(+) cells) were assessed in samples from women after SAs (n = 20) and from women with normally progressing pregnancies (NPs) (n = 27) using flow cytometry and immunohistoche­mistry. Further measured was the DPIV activity and concentrations of Th1 (interferon-gamma and tumour necrosis factor-alpha), Th2 [interleukin-4 (IL-4), IL-10] and Th3 (transforming growth factor-beta2) cytokines in serum samples. No difference was found in the number of CD4(+), CD8(+), CD26(+), CD26(+)/CD4(+) or CD8(+)/CD26(+) blood cells between NP and SA patients. No differences in the Th1, Th2 or Th3 cytokine levels could be observed between both groups. However, the percentages of decidual CD26(+) lymphocytes as well as the number of decidual Th1 cells were significantly higher in SA samples compared to samples from patients with NP.

Dipeptidyl Peptidase IV and Skin

DPIV expression on keratinocytes, in both normal and pathological skin, was investigated using immunohistochemical techniques. A sporadic focal DPIV positivity was found in normal skin, whereas increased expression of DPIV was observed both in cutaneous T-cell lymphomas and in inflammatory skin diseases, e.g. psoriasis, lichen planus and spongiotic dermatitis, in the basal and spinous layers. DPIV keratinocyte staining was not specific for a single disease, but seems to be associated with the presence of a reactive or neoplastic infiltrate in the epidermis. It was proposed that the DPIV molecule might function as a keratinocyte activation antigen.

Using flow cytometry, RT-PCR, and specific enzymatic activity assays, expression of DPIV mRNA and CD26 antigen were shown on primary keratinocyte strains and on the HaCaT keratinocyte cell line. The synthetic DPIV inhibitors Lys[Z(NO2)]-thiazolidide and -pyrrolidide suppress the DNA-synthesis of these cells in a dose-dependent manner. These data demonstrate that CD26 is also involved in the regulation of DNA synthesis of keratinocytes and that the enzymatic activity is required for mediating these effects.

There is a significant decrease (P<0.05) of DPIV/CD26 expression on CD8+ T cells in both psoriasis (7.7%+/-3.3, mean and SD, n=30) and atopic dermatitis patients (7.9%+/-3.7, mean and SD, n=15) compared to the control population (11.58%+/-5.0, mean and SD, n=17). However, there was no correlation to disease. Since DPIV/CD26 can be regarded as an anti-inflammatory principle the decreased expression in psoriasis and atopic dermatitis patients may lead to a dysbalance in favour of pro-inflammatory mediators in both clinical conditions.

Dipeptidyl Peptidase IV and Procalcitonin Truncation

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3–116), is present in the plasma of these patients. It is hypothesized that PCT(3–116) is the result of the hydrolysis of PCT(1–116) by soluble DPIV of the blood plasma or by DPIV expressed on the surface of cells.

Dipeptidyl Peptidase IV and Endothelial Wounding

DPIV‘s expression is stimulated by endothelial wounding and its enzymatic activity is required for NPY-mediated chemotaxis.

Dipeptidyl Peptidase IV and nasal mucosa

Low activity of DPIV was associated with high density of inflammatory cells in the mucosa of patients suffering from chronic rhinosinusitis. The regressive correlation was statistically significant (p < 0.001). Low level DPIV activity is associated with inflammation of the nasal mucosa. This enzyme may be involved in the pathophysiological mechanism of nasal hyperreactivity and chronic rhinosinusitis.

Dipeptidyl Peptidase IV and Cornea

In contrast to the normal cornea where DPIV activity was absint, and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPIV, which was associated with high DPIV activity in the tear fluid.

Dipeptidyl Peptidase IV in Circulation

In blood, DPIV is predominantly present in a soluble form in plasma/serum and as an activation antigen on the membrane of lymphocytes (CD26). Serum dipeptidyl peptidase IV activity was found to correlates with the T-cell CD26 antigen.

481 healthy subjects aged between 19 and 61 years were studied and the mean DPIV activity regardless of gender and age was determined to be 25,9U/L, range 12,5–42,0 U/L. A multiple regression model shows that DPIV activity decreases significantly with age. The activity in women is slightly lower than in men. An important association with liver, muscle and lipid metabolism-related parameters was observed. In this model, no significant contribution of body mass index, blood pressure or hormone therapy could be stated.

Diabetes

In a recent study, plasma DPIV activity in the fasting and the postprandial state in type-2 diabetic patients and control subjects was determined. Mean fasting plasma DPIV activity (expressed as degradation of GLP-1) was significantly higher in the diabetic group compared with the control subjects (67.5 +/- 1.9 vs 56.8 +/- 2.2 fmol GLP-1/h (mean +/- s.e.m.); P=0.001). In the type-2 diabetic patients, DPIV activity was positively correlated to fasting plasma glucose (FPG) and HbAlc and negatively to the duration of diabetes and age of the patients. No postprandial changes were seen in plasma DPIV activity in any of the groups. Plasma DPIV activity increases in the fasting state and is positively correlated to FPG and HbAlc levels, but plasma DPIV activity is not altered following meal ingestion and acute changes in plasma glucose.

Metformin and pioglitazone significantly (P<0.05) reduced serum DPIV activity and glycosylated hemoglobin in Zucker diabetic rats. Glyburide did not lower DPIV activity or glycated hemoglobin. Regression analysis showed serum DPIV activity correlated with glycated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPIV activity in vitro, indicating these are not competitive DPIV inhibitors. It was proposed that the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPIV activity results from reduced DPIV secretion.

Autoimmune Diseases

Antigen-induction of experimental arthritis (AIA) led to reduced plasma DPIV activity. In DPIV/CD26-deficient mice, the severity of AIA was increased as assessed by enhanced technetium uptake and by increased histological parameters of inflammation (synovial thickness and exudate). DPIV/CD26– deficient mice exhibited increased levels of circulating active stroma cellderived factor-1 (SDF-1), associated with increased numbers of SDF-1 receptor (CXCR4)-positive cells infiltrating arthritic joints.

In a clinical study, plasma levels of DPIV/CD26 from rheumatoid arthritis patients were significantly decreased when compared to those from osteoarthritis patients and inversely correlate with C-reactive protein levels. In conclusion, decreased circulating CD26 levels in arthritis may influence DPIV/CD26-mediated regulation of the chemotactic SDF-1/CXCR4 axis.

Serum levels of sCD26 and its specific DPIV activity were significantly decreased in systemic lupus erythematosus (SLE) and were inversely correlated with SLE disease activity index score, but not with clinical variables or clinical subsets of SLE. Close correlation between sCD26/DPIV and disease activity was observed in a longitudinal study. Serum levels of DPIV/sCD26 may be involved in the pathophysiology of SLE, and appear to be useful as a new disease activity measure for SLE.

In another study, the specific activity (as opposed to concentration) of serum DPIV was compared between rheumatoid arthritis (RA) and SLE patients and found decreased only in (RA) patients, although its levels were similar to normal controls. While both RA and SLE DPIV were hypersialylated, desialylation restored the specific activity only of RA DPIV. This finding suggests that different specific glycosylation sites in the enzyme might be involved as the underlying mechanism of the decreased enzyme specific activity of RA patients. The differences in DPIV levels observed between RA and SLE patients seem to reflect a different status of T cell activation in both diseases.

Sera from patients with rheumatoid arthritis and systemic lupus erythematosus contained low levels of DPIV and high titers of anti-DPIV autoantibodies of the immunoglobulin A (IgA) and IgG classes and a correlation between the low circulating levels of DPP IV and the high titers of anti-DPIV autoantibodies of the IgA vlase was found. Since streptokinase (SK) is a potent immunogen and binds to DPIV, it was speculated that patients with autoimmune diseases showed higher DPIV autoantibody levels than healthy controls as a consequence of an abnormal immune stimulation triggered by SK released during streptococcal infections.

Cancer

Patients diagnosed with colorectal cancer have a higher DPIV levels than healthy subjects (p<0.05). Of these patients, those with metastatic colorectal disease have a significantly higher soluble DPIV level (p<0.01). It has also been found that patients with high LDH (lactatodeshi­drogenase) and CEA (carcinoembrionary antigen) levels show higher DPIV levels (p<0.01) than patients with normal levels of such carcinoma markers.

Another study revealed significant difference between DPIV levels in healthy donors (mean 559.7 +/- 125.5 microg l(-1)) and cancer patients (mean 261.7 +/- 138.1 microg l(-1)) (P< 0.001). A cut-off at 410 microg l(-1)gave 90% sensitivity with 90% specificity which means that the diagnostic efficiency of DPIV is higher than that shown by other markers, particularly in patients at early stages. Moreover, DPIV as a variable is not related with Dukes‘ stage classification, age, gender, tumour location or degree of differentiation. With a follow-up of 2 years until recurrence, preliminary data show that DPIV can be managed as a prognostic variable of early carcinoma patients.

Heart

Bradykinin and substance P have been implicated as mediators in angiotensin- converting enzyme inhibitor (ACEI)-associated angioedema. Studies investigating the metabolism of bradykinin in sera from patients with a history of ACEI-associated angioedema and controls suggest that there is a defect in a non-ACE, non-kininase I pathway of bradykinin degradation, such as DPIV pathway.

DPIV activity was significantly lower in patients with ACEI-associated angioedema (26.9 +/- 4.1 nmol/mL per min) than in normotensive controls (37.8 +/- 6.3 nmol/mL per min), in previously ACEI-exposed untreated hypertensive volunteers, or in ACEI-treated hypertensive volunteers, even after controlling for age.

Tonsillar Hypertrophy

An increased serum DPIV activity was observed in tonsillar hypertrophy patients compared with both recurrent tonsilitis patients and controls before tonsillectomy. After tonsillectomy, all activities were similar. The results suggest that serum DPIV activity may have potential as a diagnostic marker for patients with tonsillar hypertrophy.

Infants

Beta-casomorphins, opioid peptides present in mother‘s milk, are a good substrate for DPIV which is a major factor limiting the half-life of biologically active peptides. Serum DPIV activity of two groups of infants (healthy and atopic dermatitis) and contents of beta-casomorphin-5 and –7 in their mothers‘ milk were determined in a study. It was found a correlation between those two parameters in the group of children with atopic dermatitis syndromes, while no such a correlation was found in the control group.

Liver Cirrhosis

Aberrant DPIV expression was found in human liver cirrhosis, and elevated serum DPIV activity was reported in patients with primary biliary cirrhosis and chronic hepatitis C virus infection.

Significantly higher DPIV activity was found in the sera of rats with experimental liver cirrhosis (39.2 +/- 3.7; mean +/- SD) compared to phenobarbital- treated (11 +/- 4, P < 0.000002) and nontreated (10.9 +/- 0.9, P < 0.000002) rats. There was a positive correlation between DPIV activity and concentrations of aspartate aminotransferase (r = 0.73, P = 0.0001) and alanine aminotransferase (r = 0.69, P = 0.0004). The significantly higher serum DPIV activity found in experimental liver cirrhosis is in concordance with human observations. The elevation was probably not due to the enzyme induction effect of phenobarbital. In this experimental model, serum DPIV seems to be an indicator for chronic liver injury.

Elevated serum DPIV activity was also found in patients with primary biliary cirrhosis.

Choelstasis

Dipeptidyl peptidase IV activity is elevated in the urine and serum of patients with biliary atresia (BA). To clarify the role of cholestasis in the development of increased serum and urinary DPIV/CD26 activity, the mechanism of activity increase in experimentally induced cholestasis of DPIV/CD26-deficient and wild-type rats was studied. The clinical utility of serum and urinary DPIV/ CD26 activity measurements was tested in adult and pediatric patients with hepatobiliary diseases and in liver transplant recipients. The results establish CD26-associated serum DPIV activity as a novel, clinically useful marker of cholestasis and demonstrate that in contrast with alkaline phosphatase levels, DPIV levels do not change in metastatic bone disease. Additionally, DPIV activity is useful as a urinary test of cholestasis in infants who are not receiving nephrotoxic medication.

Hepatitis C

CD26 expression has been found on the surface of human hepatoma cells transfected with hepatitis C virus (HCV) c-DNA. Serum DPIV activity was significantly higher (mean = 20.89 [s 9.6]) in patients with chronic HCV infection than in healthy controls (12.39 [2.76, P < 10(-5)]). The enzyme activities significantly differed in naive HCV-positive patients (22.2 [9.89, P < 10(-5)]) and non-responders (23.28 [9.57, P < 10(-5)]) from that in the healthy controls and also from that in responders (13.69 [4.21]). Correlation was found between DPIV activity and AST (r = 0.44, P < 10(-5)), ALT (r = 0.44, P < 10(-5)), GGT (r = 0.41, P < 10(-5)) levels. Serum DPIV activity seems to be an indicator of HCV induced

HIV Infection

To determine whether the plasma DPIV levels have clinical relevance in HIV-1 infected individuals, the concentration and DPIV enzyme activity was measured. While there is no significant difference between the plasma levels of DPIV in 90 HIV-1 infected individuals and in 79 uninfected controls, specific DPIV enzyme activity was significantly decreased in HIV-1 infected individuals (P < 0.0001). Specific DPIV enzyme activity was correlated with the levels of CD4+ T cells (r = 0.247; P < 0.02), CD8+ T cells (r = 0.236; P < 0.03), and adenosine deaminase (r = 0.227; P < 0.05) and had an inverse correlation with HIV-1 RNA (Spearman‘s r = 0.474; P = 0.0012). Furthermore, recombinant DPIV enhanced the in vitro PPD-induced response of lymphocytes from HIV-1 infected individuals with decreased specific DPIV enzyme activity. These results suggest that the specific DPIV enzyme activity may contribute to the immunopathogenesis of HIV infection.

Smoking

Reduced DPIV activity in was found in healthy smokers compared with nonsmokers. In contrast, no differences in serum or BAL fluid were observed between allergic asthmatics and healthy non-smokers.

Catalog Number	Protein	Source	Size
RD172141010mU	Dipeptidyl Peptidase IV (DPIV) Human (Human placenta)		10 mU
RD172141010mU+	Dipeptidyl Peptidase IV (DPIV) Human (Human placenta)		10 x 10 mU