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1.  Biomarkers in Chronic Fatigue Syndrome: Evaluation of Natural Killer Cell Function and Dipeptidyl Peptidase IV/CD26 
PLoS ONE  2010;5(5):e10817.
Chronic Fatigue Syndrome (CFS) studies from our laboratory and others described decreased natural killer cell cytotoxicity (NKCC) and elevated proportion of lymphocytes expressing the activation marker, dipeptidyl peptidase IV (DPPIV) also known as CD26. However, neither these assays nor other laboratory tests are widely accepted for the diagnosis or prognosis of CFS. This study sought to determine if NKCC or DPPIV/CD26 have diagnostic accuracy for CFS.
Subjects included female and male CFS cases and healthy controls. NK cell function was measured with a bioassay, using K562 cells and 51Cr release. Lymphocyte associated DPPIV/CD26 was assayed by qualitative and quantitative flow cytometry. Serum DPPIV/CD26 was measured by ELISA. Analysis by receiver operating characteristic (ROC) curve assessed biomarker potential. Cytotoxic function of NK cells for 176 CFS subjects was significantly lower than in the 230 controls. According to ROC analysis, NKCC was a good predictor of CFS status. There was no significant difference in NK cell counts between cases and controls. Percent CD2+ lymphocytes (T cells and NK cells) positive for DPPIV/C26 was elevated in CFS cases, but there was a decrease in the number of molecules (rMol) of DPPIV/C26 expressed on T cells and NK cells and a decrease in the soluble form of the enzyme in serum. Analyses by ROC curves indicated that all three measurements of DPPIV/CD26 demonstrated potential as biomarkers for CFS. None of the DPPIV/C26 assays were significantly correlated with NKCC.
By ROC analysis, NKCC and three methods of measuring DPPIV/C26 examined in this study had potential as biomarkers for CFS. Of these, NKCC, %CD2+CD26+ lymphocytes and rMol CD26/CD2+ lymphocyte, required flow cytometry, fresh blood and access to a high complexity laboratory. Soluble DPPIV/C26 in serum is done with a standard ELISA assay, or with other soluble factors in a multiplex type of ELISA. Dipeptidyl peptidase IV on lymphocytes or in serum was not predictive of NKCC suggesting that these should be considered as non-redundant biomarkers. Abnormalities in DPPIV/CD26 and in NK cell function have particular relevance to the possible role of infection in the initiation and/or the persistence of CFS.
PMCID: PMC2876037  PMID: 20520837
2.  A Role for Dipeptidyl Peptidase IV in Suppressing the Malignant Phenotype of Melanocytic Cells 
Dipeptidyl peptidase IV (DPPIV) is a cell surface peptidase expressed by normal melanocytes, epithelial cells, and other cells. Malignant cells, including melanomas and carcinomas, frequently lose or alter DPPIV cell surface expression. Loss of DPPIV expression occurs during melanoma progression at a stage where transformed melanocytes become independent of exogenous growth factors for survival. Tetracycline-inducible expression vectors were constructed to express DPPIV in human melanoma cells. Reexpressing DPPIV in melanoma cells at or below levels expressed by normal melanocytes induced a profound change in phenotype that was characteristic of normal melanocytes. DPPIV 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. Suppression of tumorigenicity and reversal of a block in differentiation were dependent on serine protease activity, assessed using mutant DPPIV molecules containing serine→alanine substitutions. Surprisingly, dependence on exogenous growth factors was not dependent on serine protease activity. Reexpression of either wild-type or mutant DPPIV rescued expression of a second putative cell surface serine peptidase, fibroblast activation protein α, which can form a heterodimer with DPPIV. This observation suggests that rescue of fibroblast activation protein α may play a role in regulating growth of melanocytic cells. These results support the view that downregulation of DPPIV is an important early event in the pathogenesis of melanoma.
PMCID: PMC2195594  PMID: 10430620
melanoma; fibroblast activating protein α; serine protease; tumorigenicity; differentiation
3.  Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells 
Virology Journal  2010;7:267.
Dipeptidyl peptidase IV (DPPIV) also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1) is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat) and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system.
The HIV1-Tat protein (Tat-BRU) co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein.
We show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.
PMCID: PMC2967539  PMID: 20942971
4.  Suppression of neuroblastoma growth by dipeptidyl peptidase IV: Relevance of chemokine regulation and caspase activation 
Oncogene  2008;28(4):479-491.
Imbalanced protease expression and activities may contribute to the development of cancers including neuroblastoma. Neuroblastoma is a fatal childhood cancer of the sympathetic nervous system that frequently overexpresses mitogenic peptides, chemokines and their receptors. Dipeptidyl peptidase IV (DPPIV), a cell surface serine protease, inactivates or degrades some of these bioactive peptides and chemokines, thereby regulating cell proliferation and survival. Our studies show that DPPIV is expressed in normal neural crest-derived structures, including superior cervical and dorsal root ganglion cells, sciatic nerve, and in adrenal glands, but its expression is greatly decreased or lost in cells derived from neuroblastoma, their malignant counterpart. Restoration of DPPIV expression in neuroblastoma cells led to their differentiation in association with increased expression of the neural marker MAP2 and decreased expression of chemokines including stromal-derived factor 1 (SDF1) and its receptor CXCR4. Furthermore, DPPIV promoted apoptosis, and inhibited SDF1 mediated in vitro cell migration and angiogenic potential. These changes were accompanied by caspase activation, and decreased levels of phospho-AKT and MMP9 activity, down stream effectors of SDF1-CXCR4 signaling. Importantly, DPPIV suppressed the tumorigenic potential of neuroblastoma cells in a xenotransplantation mouse model. These data support a potential role for DPPIV in inhibiting neuroblastoma growth and progression.
PMCID: PMC2633428  PMID: 18978811
DPPIV; Neuroblastoma; Apoptosis; Chemokine; Migration and Invasion
5.  Administration of a DPP-IV Inhibitor Enhances the Intestinal Adaptation in a Mouse Model of Short Bowel Syndrome 
Surgery  2011;150(2):217-223.
Glucagon-like peptide-2(GLP-2) induces small intestine mucosal epithelial cell (EC) proliferation; and may have benefit for patients suffering from short bowel syndrome (SBS). However, GLP-2 is rapidly inactivated in vivo by dipeptidyl peptidase IV (DPPIV). Therefore, we hypothesized that selectively inhibiting DPPIV would prolong the circulating life of GLP-2 and lead to increased intestinal adaptation after development of SBS.
8-week old C57BL/6J mice underwent a 50% proximal small bowel resection and were treated with either sitagliptin, a DPPIV-inhibitor (DPPIV-I), starting 1 day before surgery versus placebo. DPPIV-I efficacy was assessed 3 days after resection, including intestinal morphology, EC apoptosis and EC proliferation. Adaptive mechanisms were assessed with quantitative real-time PCR, and plasma bioactive GLP-2 was measured by radioimmunoassay.
Body weight loss and peripheral blood glucose levels did not change compared to SBS controls. DPPIV-I treatment led to significant increases in villus height and crypt depth. DPPIV-I treatment did not significantly change EC apoptosis rates, but significantly increased crypt EC proliferation versus placebo-SBS controls. DPPIV-I treatment markedly increased mRNA expression of β-catenin and c-myc in ileal mucosa. Plasma GLP-2 levels significantly increased(~40.9%) in DPPIV-I-SBS mice.
DPPIV- I treatment increased SBS adaptation, and may potentially be useful for SBS patients.
PMCID: PMC3148279  PMID: 21719060
6.  Dipeptidyl Peptidase IV Is a Human and Murine Neutrophil Chemorepellent 
In Dictyostelium discoideum, AprA is a secreted protein that inhibits proliferation and causes chemorepulsion of Dictyostelium cells, yet AprA has little sequence similarity to any human proteins. We found that a predicted structure of AprA has similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV is a serine protease present in extracellular fluids that cleaves peptides with a proline or alanine in the second position. In Insall chambers, DPPIV gradients below, similar to, and above the human serum DPPIV concentration cause movement of human neutrophils away from the higher concentration of DPPIV. A 1% DPPIV concentration difference between the front and back of the cell is sufficient to cause chemorepulsion. Neutrophil speed and viability are unaffected by DPPIV. DPPIV inhibitors block DPPIV-mediated chemorepulsion. In a murine model of acute respiratory distress syndrome, aspirated bleomycin induces a significant increase in the number of neutrophils in the lungs after 3 d. Oropharyngeal aspiration of DPPIV inhibits the bleomycin-induced accumulation of mouse neutrophils. These results indicate that DPPIV functions as a chemorepellent of human and mouse neutrophils, and they suggest new mechanisms to inhibit neutrophil accumulation in acute respiratory distress syndrome.
PMCID: PMC3756559  PMID: 23677473
7.  Molecular Mechanism for Connective Tissue Destruction by Dipeptidyl Aminopeptidase IV Produced by the Periodontal Pathogen Porphyromonas gingivalis  
Infection and Immunity  2005;73(5):2655-2664.
Porphyromonas gingivalis is a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion of P. gingivalis to fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.
PMCID: PMC1087322  PMID: 15845467
8.  Lymphatic-specific expression of dipeptidyl peptidase IV and its dual role in lymphatic endothelial function 
Experimental Cell Research  2008;314(16):3048-3056.
Lymphatic vessels play an important role in the maintenance of tissue fluid homeostasis and in the transport of immune cells to lymph nodes, but they also serve as the major conduit for cancer metastasis to regional lymph nodes. However, the molecular mechanisms regulating these functions are poorly understood. Based on transcriptional profiling studies of cultured human dermal lymphatic (LEC) versus blood vascular endothelial cells (BEC), we found that dipeptidyl peptidase IV (DPPIV) mRNA and protein are much more strongly expressed by cultured lymphatic endothelium than by blood vascular endothelium that only expressed low levels of DPPIV in culture. The enzymatic cleavage activity of DPPIV was significantly higher in cultured LEC than in BEC. Differential immunofluorescence analyses of human organ tissue microarrays for DPPIV and several vascular lineage-specific markers revealed that DPPIV is also specifically expressed in situ by lymphatic vessels of the skin, esophagus, small intestine, breast and ovary. Moreover, siRNA-mediated DPPIV knockdown inhibited LEC adhesion to collagen type I and to fibronectin, and also reduced cell migration and formation of tube-like structures. These results identify DPPIV as a novel lymphatic marker and mediator of lymphatic endothelial cell functions.
PMCID: PMC3398155  PMID: 18708048
Lymphatic endothelial cells; lymphangiogenesis; DPPIV; cell migration
9.  Soluble CD26/Dipeptidyl Peptidase IV Enhances the Transcription of IL-6 and TNF-α in THP-1 Cells and Monocytes 
PLoS ONE  2013;8(6):e66520.
CD26 is a 110-kDa multifunctional molecule having dipeptidyl peptidase IV (DPPIV) enzyme activity and is present on the surface of human T cells. Soluble CD26 (sCD26) exists in human blood and enhances the proliferation of peripheral T lymphocytes induced by tetanus toxoid (TT). The mechanisms by which CD26 enhances the activation of T cells and monocytes remain to be fully elucidated. In this study, we compared the stimulation of THP-1 cells and isolated human monocytes with a combination of recombinant sCD26 and lipopolysaccharide (LPS) and the stimulation of these cells with LPS alone. We found that addition of sCD26 increased TNF-α and IL-6 mRNA and protein expression and enhanced ERK1/2 levels in the cytosol as well as c-Fos, NF-κB p50, NF-κB p65, and CUX1 levels in the nuclei of these cells. On the other hand, the selective DPPIV inhibitor sitagliptin inhibited the increase in TNF-α mRNA and protein expression as well as the increase in ERK, c-Fos, NF-κB p50, NF-κB p65, and CUX1 levels. However, it did not inhibit the increase in IL-6 mRNA and protein expression. We then demonstrated that sCD26 enhanced binding of transcription factors to the TNF- and IL-6 promoters and used reporter assays to demonstrate that transcription factor binding enhanced promoter activity. Once again, we observed differential activities at the TNF- and IL-6 promoters. Finally, we demonstrated that CUX-1 overexpression enhanced TNF- production on sCD26/LPS stimulation, while CUX-1 depletion had no effect. Neither CUX-1 overexpression nor CUX-1 depletion had an effect on IL-6 stimulation. These results are discussed in the context of a model that describes the mechanisms by which stimulation of monocytic cells by sCD26 and LPS leads to elevation of TNF- and IL-6 expression. CUX-1 is identified as a new transcription factor that differently regulates the activities of the TNF- and IL-6 promoters.
PMCID: PMC3689814  PMID: 23805228
10.  Effect of CD26/dipeptidyl peptidase IV on Jurkat sensitivity to G2/M arrest induced by topoisomerase II inhibitors 
British Journal of Cancer  2003;88(3):455-462.
CD26/dipeptidyl peptidase IV (DPPIV) is a surface antigen with multiple functions, including a role in T-cell activation and the development of certain human cancers. We previously demonstrated that CD26/DPPIV enhanced sensitivity of Jurkat cells to doxorubicin. We now show that expression of CD26/DPPIV enhanced sensitivity of CD26 Jurkat transfectants to G2–M arrest mediated by the antineoplastic agent etoposide. The increased sensitivity to etoposide-induced G2–M arrest was associated with disruption of cell cycle-related events, including hyperphosphorylation of p34cdc2 kinase, change in cdc25C expression and phosphorylation, and alteration in cyclin B1 expression. CD26/DPPIV-associated enhancement of doxorubicin and etoposide-induced G2–M arrest was also observed in serum-free media, suggesting an effect of CD26 on cell-derived processes rather than serum-derived factors. Importantly, our work elucidated a potential mechanism for the enhanced susceptibility of CD26-expressing Jurkat cells to the topoisomerase II inhibitors by demonstrating that CD26/DPPIV surface expression was associated with increased topoisomerase II α levels and enhanced enzyme activity. Besides being the first to show a functional association between the multifaceted molecule CD26 and the key cellular protein topoisomerase II α, our studies provide additional evidence of a potential role for CD26 in the treatment of selected malignancies.
PMCID: PMC2747550  PMID: 12569391
CD26/DPPIV; cell cycle; G2–M; topoisomerase II α; Jurkat
11.  Dipeptidyl Peptidase 10, a Novel Prognostic Marker in Colorectal Cancer 
Yonsei Medical Journal  2013;54(6):1362-1369.
The dipeptidyl peptidase IV (DPPIV) gene family exhibits multiple functions and is involved in the pathogenesis of various diseases. It has attracted pharmaceutical interest in the areas of metabolic disorders as well as cancer. However, clinicopathologic significance of DPPIV family in colorectal cancer is not fully understood.
Materials and Methods
The clinical relevance of DPPIV and DPP10 expression was determined by immunohistochemical staining, and by assessing its clinicopathologic correlation in 383 colorectal cancer patients with known clinical outcomes.
DPPIV was not expressed in normal colon mucosa, but it showed luminal expression in 52 of the 383 colorectal cancers (13.5%). DPPIV expression in tumors was associated with right-sided location of the colon (p=0.010) and more advanced tumor stage (p=0.045). DPP10 was expressed in normal colonic mucosa, but its expression varied in primary colorectal cancer tissues. Loss of DPP10 expression was found in 11 colorectal cancers (CRCs) (2.9%), and multivariate analysis showed that loss of DPP10 expression was an independent factor for poor patient prognosis (p=0.008).
DPP10 may play a role in disease progression of colorectal cancer and loss of DPP10 expression in primary CRC is significantly associated with poor survival outcomes.
PMCID: PMC3809881  PMID: 24142639
Colorectal cancer; dipeptidyl peptidase 10; progression; prognosis
12.  CD26/dipeptidyl peptidase IV (CD26/DPPIV) is highly expressed in peripheral blood of HIV-1 exposed uninfected Female sex workers 
Virology Journal  2010;7:343.
Design of effective vaccines against the human immunodeficiency virus (HIV-1) continues to present formidable challenges. However, individuals who are exposed HIV-1 but do not get infected may reveal correlates of protection that may inform on effective vaccine design. A preliminary gene expression analysis of HIV resistant female sex workers (HIV-R) suggested a high expression CD26/DPPIV gene. Previous studies have indicated an anti-HIV effect of high CD26/DPPIV expressing cells in vitro. Similarly, high CD26/DPPIV protein levels in vivo have been shown to be a risk factor for type 2 diabetes. We carried out a study to confirm if the high CD26/DPPIV gene expression among the HIV-R were concordant with high blood protein levels and its correlation with clinical type 2 diabetes and other perturbations in the insulin signaling pathway.
A quantitative CD26/DPPIV plasma analysis from 100 HIV-R, 100 HIV infected (HIV +) and 100 HIV negative controls (HIV Neg) showed a significantly elevated CD26/DPPIV concentration among the HIV-R group (mean 1315 ng/ml) than the HIV Neg (910 ng/ml) and HIV + (870 ng/ml, p < 0.001). Similarly a FACs analysis of cell associated DPPIV (CD26) revealed a higher CD26/DPPIV expression on CD4+ T-cells derived from HIV-R than from the HIV+ (90.30% vs 80.90 p = 0.002) and HIV Neg controls (90.30% vs 82.30 p < 0.001) respectively. A further comparison of the mean fluorescent intensity (MFI) of CD26/DPPIV expression showed a higher DPP4 MFI on HIV-R CD4+ T cells (median 118 vs 91 for HIV-Neg, p = 0.0003). An evaluation for hyperglycemia, did not confirm Type 2 diabetes but an impaired fasting glucose condition (5.775 mmol/L). A follow-up quantitative PCR analysis of the insulin signaling pathway genes showed a down expression of NFκB, a central mediator of the immune response and activator of HIV-1 transcription.
HIV resistant sex workers have a high expression of CD26/DPPIV in tandem with lowered immune activation markers. This may suggest a novel role for CD26/DPPIV in protection against HIV infection in vivo.
PMCID: PMC3009705  PMID: 21108831
13.  Secreted Dipeptidyl Peptidase IV Activity in the Dimorphic Fungal Pathogen Histoplasma capsulatum▿  
Infection and Immunity  2009;77(6):2447-2454.
Dipeptidyl peptidase type IV (DppIV) enzymes are broadly distributed phylogenetically and display diverse functions, including intercellular signaling, immunomodulation, protein maturation and processing, metabolism, and nutrient acquisition. We identified a secreted proteolytic activity in Histoplasma capsulatum effective toward DppIV-specific substrates. In order to determine the gene(s) that encodes this activity, we identified two putative DPPIV homologs (HcDPPIVA and HcDPPIVB) in H. capsulatum based on a homology search with Aspergillus fumigatus DppIV. Comparative sequence analysis revealed that HcDppIVA is similar to secreted DppIV enzymes, while HcDppIVB clusters with intracellular DapB-like enzymes. Unexpectedly, silencing of HcDPPIVA by RNA interference (RNAi) had no effect on secreted DppIV activity and an HcDPPIVA-null deletion mutant also showed no abrogation of secreted DppIV activity. In contrast, RNAi silencing of HcDPPIVB significantly reduced the level of secreted DppIV activity. RNAi silencing of HcDPPIVB in the HcDPPIVA-null mutant had no additional effect on secreted DppIV activity, indicating that HcDPPIVA does not contribute to secreted activity. RNAi silencing of HcDPPIVB did not affect the ability to kill a murine macrophage-like cell line, RAW 264.7, indicating that this gene is not required for infection of macrophages.
PMCID: PMC2687333  PMID: 19349421
14.  Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo 
The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people.
The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens.
ELISA test showed significantly (p < 0.0000004 and p < 0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls.
Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alphaRI (CD89) positive cells was determined in melanoma patients (p < 0.002 and p < 0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression.
Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p < 0.0007 and p < 0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16 + CD56+) natural killer (NK) cells (p < 0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p < 0.0007 and p < 0.05 respectively).
Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response.
Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.
PMCID: PMC3457868  PMID: 22834951
Melanin; Tyrosinase; Melanoma; Vitiligo; Anti-tyrosinase antibodies; Anti-melanin antibodies; CD16+ CD56+; CD89+
15.  Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects 
Oncotarget  2013;4(12):2487-2501.
Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.
PMCID: PMC3926843  PMID: 24318733
Neuropeptide Y; Ewing sarcoma; hypoxia; cancer stem cells; angiogenesis
16.  Expression of ectopeptidases in scleroderma. 
Annals of the Rheumatic Diseases  1995;54(2):111-116.
OBJECTIVES--To examine the expression and concentrations of three ectopeptidases likely to be involved in regulating the functional levels of adhesion molecules and the turnover of connective tissue components, in patients with scleroderma (systemic sclerosis) (SSc) and in normal individuals. METHODS--Monoclonal antibodies against these antigens were used for immunoperoxidase staining of cryostat skin sections and for flow cytometric (fluorescence activated cell sorter) analysis of cultured dermal fibroblasts grown from SSc patients and normal controls. RESULTS--Although neutral endopeptidase-24.11 (NEP) (CD10) was not detected in either SSc or normal skin, aminopeptidase N (APN) (CD13) and dipeptidyl peptidase IV (DPPIV) (CD26) were both readily visualised. However, DPPIV appeared to be present in smaller concentrations in the SSc biopsy specimens. Moreover, while fibroblasts grown in vitro from both SSc and normal skin also had similar concentrations of APN, the expression of DPPIV in the cultured SSc cells was found to be very much less than that present in the normal fibroblasts. It is noteworthy that NEP, which was not detected in the tissue sections, was nevertheless readily detected in fibroblasts in culture. CONCLUSIONS--These results show that a number of cell surface proteases are expressed by dermal fibroblasts both in vivo and in vitro, and it is suggested that the marked downregulation of DPPIV in SSc could be at least partly responsible for the increased concentrations of adhesion molecules and matrix proteins associated with the molecular pathology of this disease.
PMCID: PMC1005532  PMID: 7702397
17.  Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning 
Experimental Hematology  2012;40(2):97-106.
In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1α plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV (DPPIV; DPP4) known as CD26, which cleaves dipeptides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneficial effect on hematopoietic stem cell engraftment in various but very specific experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic (i.e., CD45-congenic) and allogeneic (i.e., Balb/c to B6) BMT (1 Gy total body irradiation, 10–15 × 106 unseparated BM cells/mouse). Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up >20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (>98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting.
PMCID: PMC3265670  PMID: 22085453
18.  Characterization of the Prolyl Dipeptidyl Peptidase Gene (dppIV) from the Koji Mold Aspergillus oryzae 
Applied and Environmental Microbiology  1998;64(12):4809-4815.
The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an A. oryzae library by using the A. fumigatus dppIV gene as a probe. Reverse transcriptase PCR experiments showed that the A. oryzae dppIV gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.
PMCID: PMC90926  PMID: 9835566
19.  Dipeptidyl peptidase IV activity and/or structure homologs: Contributing factors in the pathogenesis of rheumatoid arthritis? 
Arthritis Research & Therapy  2005;7(6):253-269.
Several of the proinflammatory peptides involved in rheumatoid arthritis pathogenesis, including peptides induced downstream of tumor necrosis factor-α as well as the monocyte/T cell-attracting chemokines RANTES and stromal cell-derived factor (SDF)-1α and the neuropeptides vasoactive intestinal peptide (VIP) and substance P, have their biological half-lives controlled by dipeptidyl peptidase IV (DPPIV). Proteolysis by DPPIV regulates not only the half-life but also receptor preference and downstream signaling. In this article, we examine the role of DPPIV homologs, including CD26, the canonical DPPIV, and their substrates in the pathogenesis of rheumatoid arthritis. The differing specific activities of the DPPIV family members and their differential inhibitor response provide new insights into therapeutic design.
PMCID: PMC1297595  PMID: 16277701
20.  Increase of CD26/Dipeptidyl Peptidase IV Expression on Human Gingival Fibroblasts upon Stimulation with Cytokines and Bacterial Components 
Infection and Immunity  1999;67(12):6225-6233.
CD26/dipeptidyl peptidase IV (DPPIV) is a cell surface ectoenzyme which participates in immune and inflammatory reactions. We found that CD26 was only partially expressed on human fibroblasts from periodontal tissues, whereas fibroblasts from lung and skin expressed CD26 constitutively as revealed by flow cytometry. We examined the possible upregulation of CD26 expression on human gingival fibroblasts in response to various stimulants. Interleukin-1α (IL-1α); tumor necrosis factor alpha; gamma interferon; lipopolysaccharide from Porphyromonas gingivalis, Prevotella intermedia, and Escherichia coli; and Prevotella glycoprotein augmented CD26 expression on gingival fibroblasts. Among the stimulants, IL-1α exhibited the most potent activity. Enzymatic activity of CD26 induced by IL-1α on fibroblasts was determined colorimetrically in terms of Gly-Pro hydrolysis of a synthetic chromogenic substrate, Gly-Pro p-nitroanilide. Among various inhibitors tested, diprotin A and phenylmethylsulfonyl fluoride inhibited the enzymatic activity, suggesting that the enzyme induced by IL-1α was DPPIV. The upregulation of CD26 mRNA expression upon stimulation with IL-1α was also revealed by a quantitative reverse transcription-PCR assay. In the kinetic experiment, 48 h and several days were required for maximum CD26 mRNA accumulation and CD26 molecule expression on the cell surface, respectively. The addition of cycloheximide at 2 h before IL-1α stimulation almost completely inhibited the accumulation of CD26 mRNA. These results suggested that induction of CD26 on human gingival fibroblasts is regulated at the transcriptional level and is also dependent on a de novo-synthesized protein factor(s).
PMCID: PMC97023  PMID: 10569731
21.  T-cell activation via CD26 and caveolin-1 in rheumatoid synovium 
Modern Rheumatology  2006;16(1):3-13.
CD26 is a T-cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region. We previously reported that recombinant soluble CD26 enhances peripheral blood T-cell proliferation induced by the recall antigen tetanus toxoid (TT). Recently, we demonstrated that CD26 binds caveolin-1 on antigen-presenting cell (APC), and that residues 201–211 of CD26 along with the serine catalytic site at residue 630, which constitute a pocket structure of CD26/DPPIV, contribute to binding to caveolin-1 scaffolding domain. In addition, following CD26–caveolin-1 interaction on TT-loaded monocytes, caveolin-1 is phosphorylated, with linkage to NF-κB activation, followed by upregulation of CD86. Finally, reduced caveolin-1 expression on APC inhibits CD26-mediated CD86 upregulation and abrogates CD26 effect on TT-induced T-cell proliferation, and immunohistochemical studies revealed an infiltration of CD26+ T cells in the sublining 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 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 antigen-specific T-cell activation such as the T-cell-mediated antigen-specific response in rheumatoid arthritis.
PMCID: PMC2779407  PMID: 16622717
Caveolin-1; CD26; Memory T cell; Rheumatoid arthritis (RA); Synovial cell
22.  Thy1-positive bone marrow stem cells express liver-specific genes in vitro and can mature into hepatocytes in vivo 
Hepatology International  2007;2(1):63-71.
The bone marrow contains stem cells that have the potential to differentiate into a variety of organ-specific mature cells, including the liver and the pancreas. Recently, the origin of hepatic progenitors and hepatocytes was identified to be the bone marrow. However, evidence that describes which cells, among all bone marrow cells, differentiate into hepatocytes, has not yet been presented. Based on recent reports, hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. In particular, both hematopoietic and hepatic stem cells express the Thy1 antigen. We investigated whether rat Thy1-positive bone marrow cells express liver-specific genes in vitro, and whether transplanted Thy1 BM cells differentiate into mature hepatocytes in vivo. For collection of Thy1 cells from bone marrow, FITC-conjugated anti-Thy1.1 monoclonal antibody was used with a Fluorescence-Activated Cell Sorter system. A coculture system of 2 separate layers was used for culture of Thy bone marrow cells. Cultured Thy1 cells expressed albumin protein, which was analyzed by immunofluorescent staining. Thy1 bone marrow cells obtained from wild-type dipeptidyl peptidase IV (DPPIV(+)) male rat were directly transplanted into the injured liver of DPPIV mutant (DPPIV(−)) Fisher 344 female rats and differentiated into mature hepatocytes in recipient liver on 60 days. Donor-derived hepatocytes were confirmed by DPPIV staining and Y-chromsome in situ hybridization. Our results suggest that Thy1-positive bone marrow cells have the potential to generate liver-specific genes in vitro and can differentiate into mature hepatocytes in adult liver in vivo. Thy1-positive bone marrow stem cells may represent preexisting hepatocyte-specific stem cells.
PMCID: PMC2716869  PMID: 19669280
Thy1 antigens; Bone marrow; Stem cells; Hepatocyte
23.  Histoplasma capsulatum Encodes a Dipeptidyl Peptidase Active against the Mammalian Immunoregulatory Peptide, Substance P 
PLoS ONE  2009;4(4):e5281.
The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42°C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.
PMCID: PMC2668075  PMID: 19384411
24.  Engraftment of Syngeneic and Allogeneic Endothelial Cells, Hepatocytes and Cholangiocytes into Partially Hepatectomized Rats Previously Treated with Mitomycin C1 
Transplantation  2009;88(4):486-495.
Pretreatment with retrorsine crosslinks host hepatocyte DNA and prevents proliferation after partial hepatectomy (PH), allowing selective expansion of transplanted progenitors. Shortcomings are length of protocol and carcinogenicity of retrorsine.
This report describes a rapid liver repopulation protocol using mitomycin C (MMC) to block proliferation of rat hepatocytes in response to PH. One week post-MMC treatment, dipeptidyl peptidase IV negative (DPPIV−) host rats were given a PH followed by injection of late gestation, newborn or adult total liver isolates from DPPIV+ rats. For allogeneic transplantation, host rats received injections of anti-CD3 antibody before and after PH.
Host liver staining 2–9 weeks post-transplantation revealed well-defined donor hepatocyte colonies with strong canalicular DPPIV activity. At the same cell dose, fetal and newborn isolates produced more colonies than adult liver isolates. Hepatocyte colonies also co-expressed marker proteins characteristic of adult hepatocytes and showed polarized localization of plasma membrane proteins. Host livers contained large clusters of sinusoids lined by DPPIV+ endothelial cells co-expressing the endothelial cell marker, RECA-1 but lacked the canalicular marker leucine aminopeptidase. Colonies containing donor hepatocytes, endothelial cells and bile ducts, were also observed. Similar levels of engraftment and expansion were achieved with allogeneic liver cell isolates by using anti-CD3 antibody treatment.
The MMC transplantation model provides a rapid method for engraftment and expansion of hepatocytes, endothelial cells and cholangiocytes and should be applicable to investigations centering on the role of endothelial cells in liver regeneration and the identification and characterization of putative endothelial, hepatocyte and cholangiocyte progenitors.
PMCID: PMC2730590  PMID: 19696631
mitomycin C; partial hepatectomy; rat; dipeptidyl peptidase IV; histochemical stain
25.  A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties 
PLoS ONE  2009;4(7):e6313.
Background and Aims
Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem.
We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay.
ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant α2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions.
Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.
PMCID: PMC2708912  PMID: 19621078

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