Dipeptidyl peptidase 4 inhibitors: Applications in innate immunity?

Dipeptidyl peptidase (DPP)-4 inhibitors are a class of orally available, small molecule inhibitors that prolong the insulinotropic activity of the incretin hormone glucagon-like peptide-1 (GLP-1) and are highly effective for the treatment of Type-2 diabetes. DPP4 can also cleave several immunoregulatory peptides including chemokines. Emerging evidence continues to implicate DPP4 inhibitors as immunomodulators, with recent findings suggesting DPP4 inhibitors modify specific aspects of innate immunity. This review summarises recent insights into how DPP4 inhibitors could be implicated in endothelial, neutrophil and monocyte/macrophage mediated immunity. Additionally, this review highlights additional avenues of research with DPP4 inhibitors in the context of the COVID-19 pandemic.

Responses to thermal and salinity stress in wild and farmed Pacific oysters Crassostrea gigas.

The Pacific oyster Crassostrea gigas was introduced from Japan to many countries in the world for oyster farming, resulting in the establishment of wild populations in intertidal zones and resource competition with local faunas. This study examined physiological responses of wild oysters and farmed oysters to thermal (15°C, 25°C, 37°C and 44°C) and salinity stress (39, 50 and 60ppt). The wild oysters produced more 72kDa heat shock proteins when the temperature increased from 15°C to 25°C and 37°C and the salinity increased from 39 to 50 and 60ppt. However, the amount of 69kDa heat shock protein was similar between farmed and wild oysters when the temperature increased from 15°C to the sublethal temperature 37°C, but it was lower in wild oysters than in farmed oysters when the temperature increased from 15°C to the lethal temperature 44°C. In the tissues, wild oysters used more glycogen to promote metabolic activities by increasing the level of AEC (adenylate energy charge). The results suggest that farmed oysters might have limited ability to cope with heat stress due to low energy reserve and glycolysis activity for HSP synthesis. This study provides experimental evidence on differential responses between wild and farmed oysters to temperature and salinity changes, leading to a better understanding on the pattern of distribution for invading oyster species in the marine environment and the adaptation of marine invertebrates to the threat of climate change.

Thermal tolerance in juvenile King George whiting (Sillaginodes punctata) reduces as fish age and this reduction coincides with migration to deeper colder water.

Heat shock proteins (HSPs) are sensitive and readily produced under thermal stress in many fish species and thus serve as a useful stress bio-indicator. Two experiments were conducted to test the hypothesis that King George whiting (KGW) Sillaginodes punctata approaching sexual maturity exhibits a decrease in HSP production and that exposure to high temperatures provokes HSP production in juvenile whiting. Both adult and juvenile whiting expressed significant increases in HSP69 in response to temperature shocks of 24, 26, 28 and 30 °C. Juvenile whiting had significantly higher HSP69 than adults and expressed more HSP69 at 24 and 26 °C. No mortalities were observed in juvenile fish at 30 °C while 50% of adults suffered mortality at 30 °C. Following exposure of juveniles to 24, 26 and 28 °C, HSP69 was measured at 24, 96 and 168 h. HSP69 peaked at 96 h and returned to the 24h level after 168 h exposure. This study indicates that juveniles can cope with high temperatures better than adults, which offers a partial explanation to fish movement patterns in nature where younger fish inhabit near shore waters and then migrate to deep water towards maturation. Further, this work implies that KGW growth and recruitment can be affected by increasing temperatures due to global warming.

A pocket-sized disposable device for testing the integrity of sensation in the outpatient setting.

AIMS: To compare the Ipswich Touch Test and the VibraTip with the Neuropathy Disability Score and the vibration perception threshold for detecting the 'at-risk' foot. METHODS: We directly compared the Ipswich Touch Test and the VibraTip with both the Neuropathy Disability Score ≥ 6 and the vibration perception threshold ≥ 25 V indicating 'at-risk' feet in 83 individuals. RESULTS: The vibration perception threshold and Neuropathy Disability Score tests exhibited almost perfect agreement with each other (P < 0.001). The VibraTip and Ipswich Touch Test results were identical (P < 0.001). The VibraTip and Ipswich Touch Test results also exhibited almost perfect agreement with the vibration perception threshold (P < 0.001) and the Neuropathy Disability Score (P < 0.001). CONCLUSIONS: These two simple and efficient tests are easy to teach, reliable and can be used in any setting, and neither requires an external power source. We conclude that both the VibraTip and the Ipswich Touch Test are reliable and sensitive tests for identifying the 'high-risk' foot.

Soluble CD26 / dipeptidyl peptidase IV enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding.

Human CD26 has dipeptidyl peptidase-4 (DPP IV) enzyme activity and binds to adenosine deaminase (ADA). CD26 is costimulatory for lymphocytes and has a circulating soluble form (sCD26). DPP IV enzyme inhibition is a new successful type 2 diabetes therapy. We examined whether the ADA binding and catalytic functions of sCD26 contribute to its effects on T-cell proliferation. Wildtype soluble recombinant human CD26 (srhCD26), an enzyme inactive mutant (srhCD26E-) and an ADA non-binding mutant (srhCD26A-) were co-incubated in in vitro T-cell proliferation assays with peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA), muromonab-CD3 or Herpes simplex virus antigen (HSV Ag). Both srhCD26 and srhCD26E- enhanced PHA-induced T-cell proliferation dose-dependently in all six subjects tested. srhCD26 and srhCD26A- had no overall effect on anti-CD3-stimulated PBMC proliferation in four of five subjects. srhCD26, srhCD26E- and srhCD26A- enhanced HSV Ag induced PBMC proliferation in low responders to HSV Ag, but had no effect or inhibited proliferation in HSV-high responders. Thus, effects of soluble human CD26 on human T-cell proliferation are mechanistically independent of both the enzyme activity and the ADA-binding capability of sCD26.

Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients.

AIMS/HYPOTHESIS: The accurate detection, characterization and quantification of human diabetic neuropathy are important to define at risk patients, anticipate deterioration, and assess new therapies. Corneal confocal microscopy is a reiterative, rapid, non-invasive in vivo clinical examination technique capable of imaging corneal nerve fibres. The aim of this study was to define the ability of this technique to quantify the extent of degeneration and regeneration of corneal nerve fibres in diabetic patients with increasing neuropathic severity. METHODS: We scanned the cornea and collected images of Bowman's layer (containing a rich nerve plexus) from 18 diabetic patients and 18 age-matched control subjects. RESULTS: Corneal nerve fibre density (F(3)=9.6, p<0.0001), length (F(3)=23.8, p<0.0001), and branch density (F(3)=13.9, p<0.0001) were reduced in diabetic patients compared with control subjects, with a tendency for greater reduction in these measures with increasing severity of neuropathy. CONCLUSION/INTERPRETATION: Corneal confocal microscopy is a rapid, non-invasive in vivo clinical examination technique which accurately defines the extent of corneal nerve damage and repair and acts as a surrogate measure of somatic neuropathy in diabetic patients. It could represent an advance to define the severity of neuropathy and expedite assessment of therapeutic efficacy in clinical trials of human diabetic neuropathy.

The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort.

AIMS: To determine the incidence of, and clinically relevant risk factors for, new foot ulceration in a large cohort of diabetic patients in the community healthcare setting. METHODS: Diabetic patients (n = 9710) underwent foot screening in six districts of North-west England in various healthcare settings. All were assessed at baseline for demographic information, medical and social history, neuropathy symptom score, neuropathy disability score, cutaneous pressure perception (insensitivity to the 10 g monofilament), foot deformities, and peripheral pulses. Two years later, patients were followed up via postal questionnaire to determine the incidence of new foot ulcers. Cox's proportional hazards regression analysis was used to determine the independent, relative risk of baseline variables for new foot ulceration. RESULTS: New foot ulcers occurred in 291/6613 patients who completed and returned their 2-year follow-up questionnaire (2.2% average annual incidence). The following factors were independently related to new foot ulcer risk: ulcer present at baseline (relative risk (95% confidence interval)) 5.32 (3.71-7.64), past history of ulcer 3.05 (2.16-4.31), abnormal neuropathy disability score (> or = 6/10) 2.32 (1.61-3.35), any previous podiatry attendance 2.19 (1.50-3.20), insensitivity to the 10 g monofilament 1.80 (1.36-2.39), reduced pulses 1.80 (1.40-2.32), foot deformities 1.57 (1.22-2.02), abnormal ankle reflexes 1.55 (1.01-2.36) and age 0.99 (0.98-1.00). CONCLUSIONS: More than 2% of community-based diabetic patients develop new foot ulcers each year. The neuropathy disability score, 10 g monofilament and palpation of foot pulses are recommended as screening tools in general practice.

A comparison of the Neuropen against standard quantitative sensory-threshold measures for assessing peripheral nerve function.

AIMS: To investigate the efficacy of the Neuropen, a new clinical device that assesses both pain and pressure perception, to evaluate peripheral nerve function in diabetic patients compared with standard clinical testing methods. METHODS: Peripheral nerve function was assessed in 124 diabetic patients attending a multidisciplinary diabetes clinic, using (i) the modified Neuropathy Disability Score (NDS), derived from assessment of vibration, pinprick (pain), temperature sensation plus ankle reflexes, and (ii) vibration perception threshold (VPT) measured at the hallux. Patients were stratified into various neuropathic groups according to their NDS score. In addition, nerve function was assessed using the Neuropen, a device combining a 10-g monofilament and a weighted Neurotip. Inability to feel sensation on either or both feet for each test of the Neuropen (i.e. the monofilament or the Neurotip) was defined as an abnormal response. RESULTS: The sensitivity of the Neuropen to detect neuropathy compared with the NDS (score > or = 6/10) was high using either an abnormal monofilament response (87.8%), an abnormal Neurotip response (91.8%) or a combination of the two (82.0%). Neuropen specificity improved, however, when the combination of abnormal monofilament and abnormal Neurotip responses was used (68%), rather than the individual tests (57% and 41%, respectively). CONCLUSIONS: The Neuropen is a sensitive device for assessing nerve function and may provide an inexpensive alternative screening method to identify patients with moderate to severe neuropathy.

Risk of diabetes-related amputation in South Asians vs. Europeans in the UK.

AIMS: To compare the risk of and risk factors for diabetes-related amputation in South Asians and Europeans. METHODS: This was a population-based case control study based in the health districts of Bolton, Oldham and Central Manchester in the UK. Cases with diabetes-related amputation performed between 1992 and 1997 (n = 172) and controls with diabetes and no amputation (n = 376) were selected from the primary care-based North-west Diabetes Foot Study database. Risk factor data were also collected. RESULTS: Age at diagnosis adjusted odds ratio (OR) of amputation in South Asians compared with Europeans was 0.26, 95% confidence interval (CI) 0.11-0.65, P = 0.004. In the control population, South Asians were less likely than Europeans to have peripheral vascular disease (PVD) (9% vs. 24%, P = 0.02), neuropathy (30% vs. 54%, P = 0.003), and less likely to have ever been smokers (31% vs. 57%, P = 0.03). When these factors were added to the model, the OR was attenuated to 0.84, 95% CI 0.23-3.08, P = 0.8. CONCLUSIONS: South Asians with diabetes have about a quarter of the risk of amputation of Europeans. This is mostly explained by low rates of PVD and neuropathy in South Asians, in part associated with low rates of smoking. The reasons for the South Asian protection from both PVD and neuropathy deserve further exploration.

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND: Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM: To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS: Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Peripheral vascular and nerve function associated with lower limb amputation in people with and without diabetes.

Multiple factors, including peripheral vascular disease and neuropathy, contribute to the development and perpetuation of complications of the lower extremities in diabetes. The main aim of the present study was to assess the peripheral vascular and nerve status of diabetic and non-diabetic subjects that had undergone lower limb amputation. Various non-invasive tests of peripheral vascular and nerve function were carried out on subjects who had undergone unilateral lower limb amputation and were now attending a Rehabilitation Centre. The control group (n=23), the diabetic amputee group (n=64) and the non-diabetic amputee group (n=32) were age-matched. Only the diabetic amputee group had evidence of medial arterial calcification. Transcutaneous oxygen levels were significantly lower in the diabetic amputee group (median 43 mmHg; interquartile range 33-49 mmHg) than in the control (59; 56-74 mmHg) and non-diabetic amputee (57; 43-65 mmHg) groups (control compared with diabetic amputee group, P<0.001; diabetic amputee compared with non-diabetic amputee group, P<0.01). The same trend was found for carbon dioxide levels in the skin [mmHg: diabetic amputees, 25 (21-37); controls, 38 (32-42); non-diabetic amputee, 34 (31-39)] (control compared with diabetic amputee, P<0.01; diabetic amputee compared with non-diabetic amputee, P<0.05). Vibration and pressure perception measurements (which assess Abeta nerve fibre function) showed that both the diabetic amputee and non-diabetic amputee subjects had significantly greater impairment than the controls. However, measures of Aalpha and C nerve fibre function were abnormal only in the diabetic amputee group. Thus the peripheral vascular and nerve functions of age-matched diabetic and non-diabetic subjects having undergone lower limb amputation show specific differences, with non-diabetic amputees exhibiting signs of neuropathy. This indicates that factors characteristic of diabetes (such as hyperglycaemia and non-enzymic glycation) are associated with calcification, lower oxygen and carbon dioxide levels in the skin, and abnormal Aalpha and C nerve fibre function.

A foot care program for diabetic unilateral lower-limb amputees.

OBJECTIVE: To assess the efficacy of a specialist foot care program designed to prevent a second amputation and to assess peripheral vascular disease (PVD) and peripheral neuropathy in diabetic unilateral lower-limb amputees. RESEARCH DESIGN AND METHODS: Investigations were carried out in 143 diabetic lower-limb unilateral amputees referred to a subregional rehabilitation center for prosthetic care from a catchment area of approximately 3 million people. Peripheral vascular and nerve assessment, education, and podiatry were provided for each patient. RESULTS: For the patients referred to the foot care program, there were no baseline differences between the patients who proceeded to a bilateral amputation (n = 22) and those who remained as unilateral amputees (n = 121) in their level of foot care knowledge and mean neuropathy scores. Mean ankle-brachial pressure index was significantly lower for the bilateral amputees (0.75 +/- 0.04) compared with the unilateral amputees (0.90 +/- 0.03, mean +/- SEM, P < 0.05), but there was no difference in the level of oxygen in the skin. However, the level of carbon dioxide was significantly lower in patients with bilateral amputation (24.21 +/- 2.16 vs. 31.20 +/- 0.85 mmHg, P < 0.03). Overall, the establishment of a specialist foot care program made no impact on contralateral limb amputation (22 of 143, 15.4%) compared with matched patients without the program (21 of 148, 14%) over a 2-year outcome period for each patient. CONCLUSIONS: PVD is more closely associated with diabetic bilateral amputation than neuropathy or level of foot care knowledge. Preventative foot care programs for diabetic unilateral amputees should therefore place greater emphasis on peripheral vascular assessment to identify patients at risk and on the development of timely intervention strategies.

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.

Relating structure to function in the beta-propeller domain of dipeptidyl peptidase IV. Point mutations that influence adenosine deaminase binding, antibody binding and enzyme activity.

Point mutations in human CD26/DP IV were analysed for adenosine deaminase (ADA) binding, monoclonal antibody (mAb) binding and DP IV enzyme activity. Point mutations at either Leu294 or Val341 ablated ADA binding. Binding by mAbs that inhibit ADA binding was found to involve both Leu340 to Arg343 and Thr440/Lys441. Glu205 and Glu206 were found to be essential for enzyme activity. All residues of interest were mapped onto a model of the beta-propeller domain of DP IV. These data led us to suggest that in DP IV and related peptidases ligand and antibody binding sites are non-linear and that enzyme activity depends on charged sidechains that surround the entrance to the central tunnel of the beta-propeller.

Post-proline-cleaving peptidases having DP IV like enzyme activity. Post-proline peptidases.

DP IV has been studied extensively in disease and in the immune system by the use of enzyme assays which detect hydrolysis of Gly-Pro or Ala-Pro substrate. In addition many studies have used inhibitors of DP IV enzyme activity. The characterisation of a novel DP IV like protein, DPP4R, and of other proteases which have a substrate specificity similar to DP IV or that bind DP IV inhibitors suggests that these studies require further evaluation.

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance.

The hallmarks of chronic liver diseases are chronic inflammation, cellular damage, regeneration and fibrosis. An appreciation of intrahepatic molecular expression patterns in normal and diseased liver provides clues for understanding pathogenic pathways whilst studies of the structure and function of molecules implicated in liver disease provide insights into their potential as therapeutic targets. We have examined the expression, function, molecular structure and structure-function relationships of type IV dipeptidyl aminopeptidases. In particular, the roles of CD26/DPPIV in T-cell proliferation and chemotaxis and of fibroblast activation protein in human cirrhosis are discussed. We have investigated the pathogenesis of liver disease by characterising patterns of cytokine and growth factor expression in experimental and human cirrhosis. We have quite recently expanded this approach to use differential gene expression analyses to elucidate overall pathways of gene activation and suppression in human cirrhosis. In addition, our detailed molecular and cellular studies of the mechanisms of spontaneous liver transplant tolerance have generated novel insights into this process. This review touches on these diverse aspects of liver function and disease.

Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain.

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.

Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity.

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.