Concentration strategies for spiked and naturally present biomarkers in non-invasively collected first-void urine.

BACKGROUND: First-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation. METHODS: FVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured. RESULTS: The findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed. CONCLUSIONS: With this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.

Incidence and clinical and microbiological features of invasive and probable invasive streptococcal group A infections in children and adults in the Brussels-Capital Region, 2005-2020.

Assess the incidence, risk factors, clinical and microbiological features, and outcome of both probable invasive and invasive group A Streptococcus (GAS) infections in children and adults in the BrusselsCapital Region between 2005 and 2020. A retrospective, multicentric study was performed in three university hospitals in Brussels. Patients were identified through the centralized laboratory information system. Epidemiological and clinical data were collected from patients' hospital records. A total of 467 cases were identified. Incidence has increased from 2.1 to 10.9/100,000 inhabitants between 2009 and 2019 in non-homeless adults while it was above 100/100,000 on homeless in years with available denominators. Most of GAS were isolated from blood (43.6%), and the most common clinical presentation was skin and soft tissue infections (42.8%). A third of all the patients needed surgery, a quarter was admitted to the intensive care unit, and 10% of the adult patients died. Wounds and chickenpox disease were the main risk factors for children. Tobacco, alcohol abuse, wounds or chronic skin lesion, being homeless, and diabetes were identified as major predisposing factors for adults. The most common emm clusters were D4, E4, and AC3; 64% of the isolates were theoretically covered by the 30-valent M-protein vaccine. The burden of invasive and probable invasive GAS infections is on the rise in the studied adult population. We identified potential interventions that could contribute to decrease this burden: appropriate care of wounds, specifically among homeless and patients with risk factors such as diabetes and systematic chickenpox vaccination for children.

Is Polyomavirus-Associated Nephropathy More Common in Kidney Transplant Recipients Exposed to Valganciclovir? A Retrospective Single Center Analysis.

BACKGROUND: BK polyomavirus-associated nephropathy (PVAN) is a frequent complication in the early phase after kidney transplantation. The most important risk factor for PVAN is the intensity of immunosuppression. A recent study suggests that exposure to valganciclovir (VGC) could also be a risk factor. METHODS: We performed a retrospective, single-center study to investigate the effect of valganciclovir exposure on the risk for PVAN during the first 100 days post transplant. Cytomegalovirus (CMV) seronegative recipients of a CMV seropositive donor kidney received VGC prophylaxis, whereas CMV seropositive recipients were managed by a pre-emptive CMV strategy. Cox regression analysis was used to identify risk factors for PVAN development with VGC treatment and strength of immunosuppressive therapy as time-dependent variables. RESULTS: A total of 211 adults who received a kidney transplant between 2014 and 2019 were included. Eighteen (9%) developed PVAN. Multivariate regression analysis showed that women have a lower risk of developing PVAN (hazard ratio [HR] 0.08 (confidence interval [CI] 0.01-0.58), P = .013), whereas age was associated with an increased risk for PVAN (HR 1.04 for every additional year [CI 1.00-1.08], P = .029). There was a trend toward a lower risk of PVAN for patients on reduced immunosuppressive therapy (HR 0.44 [CI 0.15-1.24], P = .12). VGC use was not associated with the risk for PVAN (HR 0.99 [CI 0.35-2.78], P = .98). CONCLUSIONS: In our study, VGC exposure was not associated with the risk for PVAN. Our study is the first to reassess in depth the hypothesis that VGC treatment increases the risk of PVAN. The unique strength of this study is the correction for the degree of immunosuppression and the statistical use of time-dependent covariates. This methodological approach can provide a foundation for further studies needed to confirm our findings.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

Association between maternal cervicovaginal swab positivity for Ureaplasma spp. or other microorganisms and neonatal respiratory outcome and mortality.

OBJECTIVE: We investigated the association between maternal cervicovaginal cultures, its antibiotic treatment, and neonatal outcome. STUDY DESIGN: This retrospective cohort study enrolled 480 neonates born prior to 32 weeks' gestation. They were divided into groups according to maternal cervicovaginal culture results. Multivariate logistic regression analysis was used to predict neonatal outcome based on maternal culture results, adjusted for perinatal risk factors and neonatal morbidities. RESULT: Maternal cervicovaginal Ureaplasma colonization was independently associated with bronchopulmonary dysplasia at 36 weeks (BPD) (OR 8.34; 95% CI 1.21-57.45). In neonates with and without maternal cervicovaginal Ureaplasma colonization BPD occurred in 12.3% and 3.8%, respectively. Maternal colonization with other microorganisms was associated with a higher neonatal mortality (p = 0.002), lower gestational age (p = 0.026), and birth weight (p = 0.036). CONCLUSIONS: This study underscores the role of the maternal cervicovaginal microbiome as a predictor of neonatal outcome. Cervicovaginal Ureaplasma colonization seems not to be an innocent bystander in the multifactorial etiology of BPD.

Horizontally acquired papGII-containing pathogenicity islands underlie the emergence of invasive uropathogenic Escherichia coli lineages.

Escherichia coli is the leading cause of urinary tract infection, one of the most common bacterial infections in humans. Despite this, a genomic perspective is lacking regarding the phylogenetic distribution of isolates associated with different clinical syndromes. Here, we present a large-scale phylogenomic analysis of a spatiotemporally and clinically diverse set of 907 E. coli isolates, including 722 uropathogenic E. coli (UPEC) isolates. A genome-wide association approach identifies the (P-fimbriae-encoding) papGII locus as the key feature distinguishing invasive UPEC, defined as isolates associated with severe UTI, i.e., kidney infection (pyelonephritis) or urinary-source bacteremia, from non-invasive UPEC, defined as isolates associated with asymptomatic bacteriuria or bladder infection (cystitis). Within the E. coli population, distinct invasive UPEC lineages emerged through repeated horizontal acquisition of diverse papGII-containing pathogenicity islands. Our findings elucidate the molecular determinants of severe UTI and have implications for the early detection of this pathogen.

Comparison of a VLP-based and GST-L1-based multiplex immunoassay to detect vaccine-induced HPV-specific antibodies in first-void urine.

Vaccine-induced human papillomavirus (HPV) antibodies originating from cervicovaginal secretions were recently shown to be detectable in first-void (FV) urine. This presents a novel opportunity for noninvasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. With a view towards method optimization, this study compared the measurement of HPV antibodies in FV urine using a multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) with previously reported results using a glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA). We tested 53 paired FV urine and serum samples from 19- to 26-year-old healthy women, unvaccinated (n = 17) or vaccinated with either the bivalent or quadrivalent HPV-vaccine during adolescence (n = 36). HPV6/11/16/18 antibodies were measured using M4ELISA and compared with GST-L1-MIA results. Inter-assay and inter-specimen correlations were examined using the Spearman's rank test (rs). As expected, lower HPV antibody concentrations were found in FV urine than in serum. Vaccinated women had significantly higher HPV6/11/16/18 antibody levels in both FV urine and serum compared with those unvaccinated (M4ELISA; FV urine P = .0003; serum P ≤ .0001). HPV antibody levels in FV urine and serum showed a significant positive correlation (M4ELISA anti-HPV6/11/16/18, rs = 0.85/0.86/0.91/0.79, P ≤ .001). Despite assay differences, there was moderate to good correlation between M4ELISA and GST-L1-MIA (FV urine anti-HPV6/11/16/18, rs = 0.86/0.83/0.89/0.53, P ≤ .0001; serum anti-HPV6/11/16/18, rs = 0.93/0.89/0.94/0.75, P ≤ .0001). FV urine HPV antibody detection is comparable with both assays, further supporting this noninvasive sampling method as a possible option for HPV vaccine assessment. Approaches to improve the sensitivity and larger studies are warranted to determine the feasibility of FV urine for vaccine-induced HPV antibody detection.

Infection and vaccine-induced HPV-specific antibodies in cervicovaginal secretions. A review of the literature.

BACKGROUND: Human papillomavirus (HPV) infects and propagates in the cervical mucosal epithelium. Hence, in addition to assessing systemic immunity, the accurate measurement of cervical immunity is important to evaluate local immune responses to HPV infection and vaccination. This review discusses studies that investigated the presence of infection and vaccine-induced HPV-specific antibodies in cervicovaginal secretions (CVS). METHODS: We searched the two main health sciences databases, PubMed and the ISI Web of Science, from the earliest dates available to March 2019. From the eligible publications, information was extracted regarding: (i) study design, (ii) the reported HPV-specific antibody concentrations in CVS (and the associated serum levels, when provided), (iii) the CVS collection method, and (iv) the immunoassays used. RESULTS: The systematic search and selection process yielded 44 articles. The evidence of HPV-specific antibodies in CVS after natural infection (26/44) and HPV vaccination (18/44) is discussed. Many studies indicate that HPV-specific antibody detection in CVS is variable but feasible with a variety of collection methods and immunoassays. Most CVS samples were collected by cervicovaginal washing or wicks, and antibody presence was mostly determined by VLP-based ELISAs. The moderate to strong correlation between vaccine-induced antibody levels in serum and in CVS indicates that HPV vaccines generate antibodies that transudate through the cervical mucosal epithelium. CONCLUSION: Although HPV-specific antibodies have lower titres in CVS than in serum samples, studies have shown that their detection in CVS is feasible. Nevertheless, the high variability of published observations and the lack of a strictly uniform, well-validated method for the collection, isolation and quantification of antibodies indicates a need for specific methods to improve and standardize the detection of HPV-specific antibodies in CVS.

Dipeptidyl peptidases in atherosclerosis: expression and role in macrophage differentiation, activation and apoptosis.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 ± 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 ± 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 ± 16 vs. 146 ± 19 pg/ml; TNFα 3.8 ± 1.0 vs. 6.6 ± 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFα and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 ± 4 vs. 7 ± 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.

Dipeptidyl peptidase 4 as a therapeutic target in ischemia/reperfusion injury.

Dipeptidyl peptidase 4 (DPP4, DPPIV, CD26, EC 3.4.14.5) was discovered more than four decades ago as a serine protease that cleaves off N-terminal dipeptides from peptide substrates. The development of potent DPP4 inhibitors during the past two decades has led to the identification of DPP4 as a target in the treatment of type 2 diabetes. The favorable effect of DPP4 inhibitors is based on prevention of the in vivo inactivation of the incretin hormone, glucagon-like peptide-1 (GLP-1) by DPP4. Apart from GLP-1, a number of other biologically active peptides are truncated by DPP4. For these peptides, the physiological relevance of their truncation has yet to be fully elucidated. Within the last 10years, DPP4 inhibitors have been employed in several animal models of lung and heart disease, in which injury was induced by an ischemic insult followed by subsequent reperfusion. In this review, we present a state-of-the-art of the ischemia/reperfusion injury (IRI)-related pharmacological actions of DPP4 substrates, including GLP-1, stromal cell-derived factor-1 alpha and vasoactive intestinal peptide. Furthermore, we discuss the large body of experimental work that now provides compelling evidence for the advantageous impact of DPP4 targeting in IRI. However, possible risks as well as underlying mechanisms are yet to be elucidated before translating these promising treatment strategies into clinical practice.

DPP4 inhibition improves functional outcome after renal ischemia-reperfusion injury.

Dipeptidyl peptidase 4 (DPP4) is an exopeptidase which modulates the function of its substrates, among which are insulin-releasing incretins. DPP4 inhibitors are currently used to improve glucose tolerance in type 2 diabetes patients. Inhibition of DPP4 exhibits protective effects on ischemia-reperfusion injury (IRI) of the heart and lung. As DPP4 and its substrates are also expressed in the kidney, we studied the effect of the DPP4 inhibitor vildagliptin on the outcome of IRI-induced acute kidney injury in rats in a model of 30-min unilateral renal ischemia, followed by contralateral nephrectomy. Saline, 1, or 10 mg/kg vildagliptin (VG1/VG10) was administered intravenously 15 min before the surgery. Animals were euthanized after 2, 12, amd 48 h of reperfusion. DPP4 inhibition resulted in a significant dose-dependent decrease in serum creatinine (1.31 ± 0.32 and 0.70 ± 0.19 mg/dl for VG1 and VG10, respectively, vs. 1.91 ± 0.28 mg/dl for controls at 12 h; P < 0.01). Tubular morphology (PAS-PCNA) revealed significantly reduced tubular necrosis at 12 h (62.1 ± 18.0 and 77.5 ± 22.0% in VG10 and saline, respectively). VG did not affect regeneration but decreased apoptosis, as shown by twofold decreased Bax/Bcl-2 mRNA expression and a threefold decrease in apoptotic bodies on terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained sections. VG treatment significantly reduced serum malondialdehyde twofold in both VG1- and VG10-treated ischemic and sham-operated animals compared with controls and also resulted in a significant decrease in mRNA expression of the proinflammatory marker CXCL10 at 2 h of reperfusion. Through a mechanism yet to be fully understood, VG treatment results in a functional protection of the kidney against IRI. This protection was associated with antiapoptotic, immunological, and antioxidative changes.

CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation.

OBJECTIVES: The CD26 antigen is a transmembrane glycoprotein that is constitutively expressed on activated lymphocytes and in pulmonary parenchyma. This molecule is also identified as dipeptidyl peptidase-4 (DPP-4) that cleaves a host of biologically active peptides. Here, we aimed to identify an important substrate of CD26/DPP-4-stromal cell-derived factor-1 (SDF-1/CXCL12)-as a key modulator for stem-cell homing together with its receptor CXCR4 in response to ischaemic injury of the lung. METHODS: Orthotopic single lung transplantation (Tx) was performed between syngeneic C57BL/6 mice. Inhibition of CD26/DPP-4 activity in recipients was achieved using vildagliptin (10 mg/kg, every 12 h) subcutaneously, and 6 h ischaemia time was applied prior to implantation. Forty-eight hours after Tx, lung histology, SDF-1 levels (enzyme-linked immunosorbent assay) in lung, spleen and plasma, and expression of the SDF-1 receptor CXCR4 in blood and lung were assessed. Homing of regenerative progenitor cells to the transplanted lung was evaluated using fluorescent-activated cell sorting. RESULTS: Compared with untreated lung transplanted mice, systemic DPP-4 inhibition of Tx recipients resulted in an increase in protein concentration of SDF-1 in plasma, spleen and lung. Concordantly, the frequency of cells bearing the SDF-1 receptor CXCR4 rose significantly in the circulation and also in the lungs of DPP-4-inhibited recipients. We found co-expression of CXCR4/CD34 in the grafts of animals treated with vildagliptin, and the stem-cell markers Flt-3 and c-kit were present on a significantly increased number of cells. The morphology of grafts from DPP-4 inhibitor-treated recipients revealed less alveolar oedema when compared with untreated recipients. CONCLUSIONS: Targeting the SDF-1-CXCR4 axis through CD26/DPP-4 inhibition increased the intragraft number of progenitor cells contributing to the recovery from ischaemia-reperfusion lung injury. Stabilization of endogenous SDF-1 is achievable and may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept.

Dipeptidyl peptidase IV inhibition improves cardiorenal function in overpacing-induced heart failure.

AIMS: Recent studies indicate that brain natriuretic peptide (BNP(1-32)) may be truncated into BNP(3-32) by dipeptidyl peptidase IV (DPP4) and that BNP(3-32) has reduced biological activities compared with BNP(1-32). We investigated if DPP4 contributes to the cardiorenal alterations and to the attenuated response to BNP seen in heart failure. METHODS AND RESULTS: Haemodynamic and renal assessment was performed in 12 pigs at baseline, 4 weeks after pacing-induced heart failure, and during BNP infusion. They were randomized to either placebo or treatment with a DPP4 inhibitor, sitagliptin. After 4 weeks of pacing, heart rate was reduced compared with baseline in the sitagliptin group (60 ± 2 vs. 95 ± 16 b.p.m., P < 0.01), and an increase in stroke volume was observed in the sitagliptin group compared with placebo (+24 ± 6% vs. -17 ± 7%, P < 0.01). Glomerular filtration rate declined at week 4 compared with baseline in the placebo group (1.3 ± 0.4 vs. 2.3 ± 0.3 mL/kg/min, P < 0.01) but remained preserved in the sitagliptin group [1.8 ± 0.2 vs. 2.0 ± 0.3 mL/kg/min, P = NS (non-significant)]. In the sitagliptin group, BNP infusion improved end-systolic elastance (68 ± 5 vs. 31 ± 4 mmHg/kg/mL, P < 0.05), ventricular-arterial coupling, and mechanical efficiency. Compared with controls (n = 6), myocardial gene expression of BNP, interleukin-6, Na(+)-Ca(2+) exchanger, and calmodulin was up-regulated in the placebo group, but not in the sitagliptin group. CONCLUSION: In pacing-induced heart failure, DPP4 inhibition preserves the glomerular filtration rate, modulates stroke volume and heart rate, and potentiates the positive inotropic effect of exogenous BNP at no energy expense.

Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning.

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 × 10(6) 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.

Method comparison of dipeptidyl peptidase IV activity assays and their application in biological samples containing reversible inhibitors.

BACKGROUND: Dipeptidyl peptidase IV (DPPIV, DPP4) is a serine protease that releases N-terminal dipeptides. It is a validated drug target for type 2 diabetes and DPPIV inhibitors are currently evaluated for other therapeutic applications. Various assays are used for DPPIV activity measurements in biological samples. Highly sensitive methods are needed to measure also very low activities in inhibited samples. METHODS: Here, the three most extensively used substrates to quantify DPPIV activity are compared using in-house methods. A luminescent kit was also included. In addition, one of the in-house fluorometric assays was elaborated for use in biological samples containing reversible DPPIV inhibitors to estimate residual DPPIV activity which is usually underestimated due to sample dilution. RESULTS: The in-house methods showed a good precision, linearity and specificity. Both fluorometric substrates had a 10-fold higher sensitivity compared to the colorimetric assay. The luminescent kit was found to be the most sensitive. CONCLUSIONS: All three in-house methods can be used to measure DPPIV activity in non-inhibited biological samples. The more sensitive fluorometric assays are recommended when sample volumes are limited or when using inhibited samples. The elaborated fluorometric method can be used to estimate the residual in vivo DPPIV activity in inhibitor treated subjects.

Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries.

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.

Inhibition of CD26/DPP IV attenuates ischemia/reperfusion injury in orthotopic mouse lung transplants: the pivotal role of vasoactive intestinal peptide.

The T cell activation Ag CD26/dipeptidylpeptidase IV (DPP IV) combines co-stimulatory and enzymatic properties. Catalytically, it functions as an exopeptidase, modulating biological activity of key chemokines and peptides. Here we investigated the effect of organ-specific inhibition of DPP IV catalytic activity on ischemia/reperfusion injury after extended ischemia in the mouse model of orthotopic single lung transplantation. C57BL/6 mice were syngeneically, transplanted, grafts were perfused and stored in Perfadex with (treated) or without (control) a DPP IV enzymatic activity inhibitor (AB192). Transplantation was performed after 18h cold ischemia time; following 2-h reperfusion, grafts were analyzed for oxygenation, thiobarbituric acid-reactive substances, histomorphology, and immunohistochemistry was performed for leukocyte Ag 6, myeloperoxidase, hemoxygenase 1, vasoactive intestinal protein (VIP), and real-time PCR for VIP. Treatment with the DPP IV inhibitor AB192 resulted in significant improvement of gas exchange, less lipid oxidation, preservation of parenchymal ultrastructure, reduced neutrophil infiltration, reduced myeloperoxidase expression, increased hemoxygenase 1 expression, pronounced expression of VIP in alveolar macrophages and increased mRNA expression of VIP. Inhibition of intragraft DPP IV catalytic activity with AB192 strikingly ameliorates ischemia/reperfusion injury after extended ischemia. Furthermore, preservation of endogenous intragraft VIP levels correlate with maintaining lung function and structural integrity.