Investigating the Clinical Validity of CUB and Zona-Pellucida-like Domain-Containing Protein 1 (CUZD1) in Malignant and Non-Malignant Human Diseases

CUB and zona pellucida-like domain-containing protein 1 (CUZD1) has been previously shown to be specifically expressed in normal pancreas and was proposed as a candidate biomarker for pancreatic related disorders. Due to the lack of specific reagents and techniques, its levels in tissues and biological fluids have not been extensively examined. We generated mouse monoclonal antibodies against recombinant CUZD1 and used them for the development of an enzyme-linked immunosorbent assay (ELISA). Analysis of various human extracts showed that CUZD1 is measured in high levels in pancreas and at much lower (but detectable) levels in several other tissues. Analysis of biological fluids showed that CUZD1 is detected exclusively in pancreatic juice. CUZD1 has been previously linked to diseases (such as pancreatitis, ovarian cancer and IBD) but it is currently unknown if the expression levels of this antigen are elevated in any of the aforementioned or other disorders. Analysis of a large number of serum samples from patients with various malignant and benign disorders showed that CUZD1 levels were elevated in patients with ovarian cysts but not ovarian cancer. CUZD1 is a pancreas-specific protein but it is unclear if its expression is elevated in malignant conditions of the pancreas. IHC staining of pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma (ACC) tissue sections revealed that CUZD1 protein was highly expressed in ACC but not in PDAC. CUZD1 is one of the targets of pancreatic autoantibodies (PABs) which have been emerged as possible biomarkers for Inflammatory Bowel Disease (IBD). Data assessing the diagnostic significance of CUZD1 autoantibodies in patients with IBD are scarce, mainly due to the lack of high throughput techniques for their detection. We developed an ELISA targeting CUZD1 autoantibodies and used it to analyze 200 serum samples from IBD patients and 129 patients assessed for various autoimmune diseases (vADs). CUZD1 autoantibodies were detected in 16% of CrD patients in 9% of UC patients and in less than 5% of patients being tested for vADs. In conclusion, this thesis encompasses the development and validation of analytical techniques targeting CUZD1 antigen and CUZD1 autoantibodies. These tools can facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology.Ph.D

Cancer immunotherapy: the beginning of the end of cancer?

Abstract These are exciting times for cancer immunotherapy. After many years of disappointing results, the tide has finally changed and immunotherapy has become a clinically validated treatment for many cancers. Immunotherapeutic strategies include cancer vaccines, oncolytic viruses, adoptive transfer of ex vivo activated T and natural killer cells, and administration of antibodies or recombinant proteins that either costimulate cells or block the so-called immune checkpoint pathways. The recent success of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), has boosted the development of this treatment modality, with the consequence that new therapeutic targets and schemes which combine various immunological agents are now being described at a breathtaking pace. In this review, we outline some of the main strategies in cancer immunotherapy (cancer vaccines, adoptive cellular immunotherapy, immune checkpoint blockade, and oncolytic viruses) and discuss the progress in the synergistic design of immune-targeting combination therapies

Biochemical and functional characterization of the human tissue kallikrein 9

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.</p

Novel immunoassays for detection of CUZD1 autoantibodies in serum of patients with inflammatory bowel diseases

AbstractBackground:Pancreatic autoantibodies (PABs) are detected in patients with inflammatory bowel disease (IBD). Their prevalence is higher in Crohn’s disease (CrD) than in ulcerative colitis (UC). Glycoprotein 2 (GP2) and, more recently, CUB and zona pellucida-like domain-containing protein 1 (CUZD1) have been identified as target autoantigens of PAB. The clinical utility of CUZD1 autoantibodies has only recently been assessed by indirect immunofluorescence (IIF) assays. In this study, we developed and validated novel immunoassays for the detection of CUZD1 autoantibodies.Methods:Recombinant CUZD1 protein was utilized as a solid-phase antigen for the development of two immunoassays for the detection of IgG and IgA CUZD1 autoantibodies. Serum samples from 100 patients with CrD, 100 patients with UC, 129 patients assessed for various autoimmune diseases (vADs) and 50 control individuals were analyzed.Results:Two immunofluorometric assays for the detection of IgG and IgA CUZD1-specific antibodies were developed. CUZD1 autoantibodies were detected in 12.5% (25/200) IBD patients, including 16% of patients with CrD and in 9% of patients with UC (CrD vs. UC, p&lt;0.05), compared with 3.1% (4/129) patients suspected of having vADs (CrD vs. ADs, p&lt;0.05; UC vs. ADs, p=0.08). CUZD1 autoantibody positivity was not found to be related to disease location, age of disease onset or disease phenotype.Conclusions:This is the first study to describe novel IgA and IgG CUZD1 autoantibody enzyme-linked immunosorbent assay. These immunoassays agree well with standard IIF techniques and can be utilized in multicenter studies to investigate the diagnostic and clinical utility of CUZD1 autoantibodies.</jats:sec

MOESM9 of A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

Additional file 9: Figure S5. KLK9 free monomer detection by the newly developed KLK9 ELISA upon the formation of serpinA3–KLK9 heterocomplexes. Mat-KLK9 (0.5 μg) was incubated either alone (control) or with different amounts of the human recombinant serpinA3 inhibitor (R&D systems) [at molar ratios (KLK9/SerpinA3): 1/0.2, 1/0.5, 1/1 and 1/2] in 50 mM Tris–HCl (pH 8.0) for 1 h at 37 °C. The samples were further diluted with 6% BSA and the assay was performed by following the described protocol (see “Methods” section). The values of the sample containing no inhibitor (control) was arbitrarily defined as 100 % recovery. The samples containing serpinA3–KLK9 complexes were expressed as % recovery of KLK9 compared to the control

Association between the renin-angiotensin system and chronic lung allograft dysfunction

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin system, elicits fibrosis in both kidney and lung. We identified six AngII-regulated proteins (Ras homolog family member B (RHOB), bone marrow stromal cell antigen 1 (BST1), lysophospholipase 1 (LYPA1), glutamine synthetase (GLNA), thrombospondin 1 (TSP1) and laminin subunit β2 (LAMB2)) that were increased in urine of patients with kidney allograft fibrosis. We hypothesised that the renin-angiotensin system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2), TSP1 and GLNA in 10 CLAD lungs and five controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (stable, acute lung allograft dysfunction (ALAD) and CLAD). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1+cells in CLADversuscontrol lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R2=0.42 and 0.57, respectively). In BAL, we noted a trend towards higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p&lt;0.05). The support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy (area under the curve (AUC) 0.86) and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the renin-angiotensin system are increased in CLAD lungs and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development