Inhibition of circulating dipeptidyl peptidase 4 activity in patients with metastatic prostate cancer.

Cancer is responsible for many deaths and is a major source of healthcare expenditures. The identification of new, non-invasive biomarkers might allow improvement of the direct diagnostic or prognostic ability of already available tools. Here, we took the innovative approach of interrogating the activity of exopeptidases in the serum of cancer patients with the aim of establishing a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease relative to cancer-free littermates. We identified the protease in question as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in the sera of patients with metastatic prostate cancer relative to patients with localized disease or healthy controls. However, no significant differences in DPP4 serum levels were observed, which established the loss of activity as the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity was the result not of post-translational modifications or allosteric changes, but instead of a low-molecular-weight inhibitor. After we adjusted for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity might be a potential blood-based indicator of the presence of metastatic cancer of prostatic origin, either by itself or, more likely, as a means to improve the sensitivity and specificity of existing markers.

Serum 25-hydroxyvitamin D deficiency; a risk factor for chronic kidney disease in ambulatory indigent patients.

OBJECTIVE: To assess whether 25-hydroxyvitamin D (25[OH]D) deficiency is a risk factor for chronic kidney disease (CKD) in ambulatory indigent patients. METHODS: Data for all serum 25(OH)D concentrations measured during 2010 in our ambulatory nondialysis-dependent patients were analyzed along with CKD-related parameters. Patients were stratified into groups based on 25(OH)D levels of <10, 10 to 19, 20 to 29, and ≥30 ng/mL. CKD was defined by estimated glomerular filtration rate (eGFR; Chronic Kidney Disease-Epidemiology Collaboration [CKD-EPI] equation) and abnormal urine protein to creatinine ratios. CKD-associated parameters included serum parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25[OH]2D), alkaline phosphatase, albumin, corrected calcium, and total CO2 levels. RESULTS: A total of 2,811 patients had 25(OH)D levels measured. Patients with 25(OH)D levels <10 ng/mL had significantly increased relative risk (RR) of an eGFR <15 mL/min/1.73 m2 (RR, 4.0), an eGFR of 15 to 29 mL/min/1.73 m2 (RR, 2.6), urine protein to creatinine ratio >3.5 g/g (RR, 5.6), and serum PTH >100 pg/mL (RR, 2.8) compared to patients with a 25(OH)D level ≥30 ng/mL. Patients with 25(OH)D levels of 10 to19 ng/mL had significantly increased RR of a urine protein to creatinine ratio >3.5 g/g (RR, 4.8) and serum PTH >100 pg/mL (RR, 1.5) compared to patients with 25(OH)D levels ≥30 ng/mL. CONCLUSION: 25(OH)D deficiency (<10 ng/mL) was associated with reduced eGFR, nephrotic-range proteinuria, and increased PTH levels in our population of ambulatory urban indigent patients.

Vitamin D deficiency in urban indigent patients in Southern California.

OBJECTIVE: To determine the prevalence of 25-hydroxy (OH) vitamin D deficiency in ambulatory and hospitalized patients from a large urban county medical center in Southern California, and assess the effects of season, ethnicity, age, location of care, and comorbidities on prevalence. METHODS: Data for all serum 25(OH)-D2 and -D3 concentrations measured during 2010, along with associated demographic characteristics and comorbidity data, were analyzed. 25(OH) D concentrations were measured using liquid chromatography-tandem mass spectrometry. RESULTS: Of 210,695 patients, serum 25(OH) D concentrations were measured for 3,276 (1.6%), 78% of whom were Hispanic, 69% female, 14% hospitalized, and 86% ambulatory. Median patient age was 54 years. Prevalence of 25(OH) D <10 ng/mL was 6.5% overall, 5.5% in Hispanics, 6.7% in Asians, 15.5% in African Americans, and 8.9% in whites. Prevalence was significantly higher in African Americans than in Hispanics (relative risk (RR): 2.79), males (RR: 2.07), hospitalized patients (RR: 4.96), and winter (RR: 1.34). Prevalence of 25(OH) D <20 ng/mL was 35% overall, 34% in Hispanics, 32% in Asians, 49% in African Americans, and 33% in whites, and was significantly higher in African Americans than Hispanics (RR: 1.45), males (RR: 1.32), hospitalized patients (RR: 2.02), and younger patients (RR: 1.21, age ≤30; 1.16, age 31-50) versus those age 51 to 70 years, and in winter (RR: 1.21). CONCLUSION: Our study estimated the prevalence of 25(OH) D deficiency and identified at-risk patient groups in Southern California; 25(OH) D deficiency should be suspected, diagnosed, and adequately treated to improve the health status in at-risk urban indigent patient populations.

A sequence-specific exopeptidase activity test (SSEAT) for "functional" biomarker discovery.

One form of functional proteomics entails profiling of genuine activities, as opposed to surrogates of activity or active "states," in a complex biological matrix: for example, tracking enzyme-catalyzed changes, in real time, ranging from simple modifications to complex anabolic or catabolic reactions. Here we present a test to compare defined exoprotease activities within individual proteomes of two or more groups of biological samples. It tracks degradation of artificial substrates, under strictly controlled conditions, using semiautomated MALDI-TOF mass spectrometric analysis of the resulting patterns. Each fragment is quantitated by comparison with double labeled, non-degradable internal standards (all-d-amino acid peptides) spiked into the samples at the same time as the substrates to reflect adsorptive and processing-related losses. The full array of metabolites is then quantitated (coefficients of variation of 6.3-14.3% over five replicates) and subjected to multivariate statistical analysis. Using this approach, we tested serum samples of 48 metastatic thyroid cancer patients and 48 healthy controls, with selected peptide substrates taken from earlier standard peptidomics screens (i.e. the "discovery" phase), and obtained class predictions with 94% sensitivity and 90% specificity without prior feature selection (24 features). The test all but eliminates reproducibility problems related to sample collection, storage, and handling as well as to possible variability in endogenous peptide precursor levels because of hemostatic alterations in cancer patients.

Retinoylserine and retinoylalanine, natural products of the moth Trichoplusia ni.

Insect cells convert vitamin A into a number of retinoids that are evolutionarily conserved with those of mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic acid peptides, N-trans-retinoylserine (1) and N-trans-retinoylalanine (2), have been isolated from a cell line of the common cabbage looper, Trichoplusia ni. These are the first examples of naturally occurring retinoic acid linked to amino acids through an amide bond; the amino acid moieties are depicted in the more common l-configuration, although the absolute configuration was not determined due to the minuscule sample amount.

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn.

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.

Comparison of antibody titers after immunization with monovalent or tetravalent KLH conjugate vaccines.

Antigens such as ganglioside GD3, neutral glycolipid Lewis(y) (Le(y)) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3-KLH, Le(y)-KLH, MUC1-KLH and MUC2-KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.