Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion.

Carboxypeptidase E (CPE), a prohormone processing enzyme is highly expressed and secreted from (neuro)endocrine tumors and gliomas, and has been implicated in cancer progression by promoting tumor growth. Our study demonstrates that secreted or exogenously applied CPE promotes survival of pheochromocytoma (PC12) and hepatocellular carcinoma (MHCC97H) cells under nutrient starvation and hypoxic conditions, but had no effect on their proliferation. CPE also reduced migration and invasion of fibrosarcoma (HT1080) cells. We show that CPE treatment mediates survival of MHCC97H cells during metabolic stress by up-regulating the expression of anti-apoptotic protein BCL-2, and other pro-survival genes, via activation of the ERK1/2 pathway.

[Detection of carboxypeptidase H specific T cells in peripheral blood of latent autoimmune diabetic patients with carboxypeptidase antibody positivity by ELISPOT assay].

OBJECTIVE: To explore the characteristics of T cell immunity in peripheral blood of patients with carboxypeptidase-H antibody (CPH-Ab). METHODS: Forty-two latent autoimmune diabetes in adults (LADA) patients with CPH-Ab(+) alone, 20 Type 2 diabetes mellitus patients (T2DM), and 22 healthy controls were selected and their peripheral blood mononuclear cells were isolated. Human recombinant carboxypeptidase (CPH) protein was expressed and further used as a stimulant in Enzyme-linked immunospot (ELISPOT) assay to detect IFN-gamma-Th1 and IL-4-Th2 cells in the 3 groups. Th1/Th2 ratios were also calculated. CPH-Ab and glutamic acid decarboxylase antibody (GAD-Ab) were determined by radioligand assay. RESULTS: Compared with healthy controls and T2DM, IFN-gamma-Th1 and IL-4-Th2 numbers did not increase significantly in CPH-Ab(+) group, nor did the Th1/Th2 ratios (P>0.05). We further divided the CPH-Ab(+) patients into a short duration group (n=22) and a long duration subgroup (n=20) according to the duration of 3 years. CPH-IL-4-T in the short duration subgroup was significantly higher than that in T2DM and healthy controls (1.8 vs. 0.2 and 0.3, both P<0.05) and we did not find any factor that was significantly correlated with the IL-4 spots number. There were not any significant differences in T cell responses to phaseolus vulgaris agglutinin (PHA) among all groups (P>0.05). CONCLUSION: CPH does not directly involve in the cellular pathological mechanism of LADA. Anti-CPH immunity may be associated with more slowly aggressive beta cell autoimmunity.

Carboxypeptidase-H autoantibodies differentiate a more latent subset of autoimmune diabetes from phenotypic type 2 diabetes among Chinese adults.

This study aimed to investigate whether carboxypeptidase-H antibody (CPH-Ab) can help identify latent autoimmune diabetes in adults (LADA). Phenotypic type 2 diabetic (T2D) patients (n= 1296) were studied for CPH Abs and autoantibodies to glutamic acid decarboxylase (GAD-Abs). CPH-Ab(+) T2D patients also underwent testing for insulinoma protein tyrosine phosphatase (IA-2A). Clinical features were compared among CPH-Ab(+), GAD-Ab(+), and Ab(-) T2D patients. Some of the antibody-positive patients were followed up for 3 years to assess beta cell function. The prevalence of CPH-Abs in T2D patients was 4.8%, significantly higher than that in controls. Double positivity was rare between CPH-Abs and GAD-Abs or IA-2A. Compared to patients with Ab(-) T2D, those with CPH-Ab(+) T2D had lower BMI, lower fasting C-peptide (FCP) levels, and more frequent ketosis, while not as much as did those with GAD-Ab(+) T2D. The mild beta cell dysfunction in patients with CPH-Ab(+) T2D was associated with their longer duration of diabetes. No marked change of C-peptide in the CPH-Ab(+) group was found during follow-up. These findings demonstrated that CPH-Abs may allow discrimination of a more latent subset of adult-onset autoimmune diabetes (LADA) whose features are intermediate between those with classic GAD-Ab(+) LADA and patients with Ab(-) T2D.

Islet autoimmunity and genetic mutations in Chinese subjects initially thought to have Type 1B diabetes.

AIMS: To explore the contribution of islet autoimmunity and genetic mutations in Chinese patients initially thought to have Type 1B diabetes. METHODS: A group of 33 Chinese patients with newly diagnosed Type 1B diabetes, were identified by the absence of autoantibodies to glutamic acid decarboxylase (GAD), IA-2, insulin, thyroid globulin or thyroid peroxidase, or high-risk HLA-DQ haplotypes. The cohort was further characterized by measurement of autoantibodies to carboxypeptidase H (CPH) and SOX13 using radioligand assays, and testing for genetic mutations associated with MODY3/MODY6 and mitochondrial diabetes. Mutations of HNF-1alpha (MODY3) and neuroD1/beta2 (MODY6) genes were screened using the single-strand conformation polymorphism (SSCP) technique and sequencing. Mitochondrial DNA mutations were analysed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Within the cohort, we found one patient with a novel mutation, R321H (CGC-->CAC) in exon 5 of the HNF-1alpha gene, one with ND1 mt3316 G-->A mutation in mitochondrial DNA, five with Ala45Thr polymorphisms in the neuroD1/beta2 gene, and two patients with autoantibodies to SOX13. CONCLUSIONS: Some of the Chinese patients originally thought to have Type 1B diabetes do have other evidence of islet autoimmunity and genetic mutations involved in the underlying aetiology. This suggests that more rigorous screening for these conditions is needed before classifying subjects as having Type 1B diabetes.

[Cross-sectional study of the relation between carboxypeptidase-H antibody and islet beta cell function in patients with latent autoimmune diabetes in adults].

OBJECTIVE: To explore the relation between carboxypeptidase-H antibody (CPH-Ab) and islet beta cell function in patients with latent autoimmune diabetes in adults (LADA) and to further confirm the diagnostic value of CPH-Ab for LADA. METHODS: Five hundred and forty-five patients who were initially diagnosed as Type 2 diabetes mellitus (T2DM) were tested with CPH-Ab and GAD-Ab by radioligand assay (RLA). T2DM patients, according to CPH-Ab and GAD-Ab status, were divided into CPH-Ab(+) group, GAD-Ab(+) group, and Ab(-) group to compare their islet beta cell function [represented by fasting C-peptide (FCP) and 2h postprandial C-peptide (2hCP)]. The relation between CPH-Ab and islet beta cell function in LADA was analyzed. RESULTS: The fasting C-peptide level in CPH-Ab(+) patients was between that of GAD-Ab(+) patients and that of Ab(-) patients (P<0.05), and the difference was still significant when the 3 groups were stratified with duration of disease (All P<0.05), but not with body mess index (all P>0.05). Corrected by concomitant variables including age, age at onset, duration of disease, and sex, the differences among the 3 groups were statistically significant (both P<0.001). Among the 3 groups FCP was lower than Ab(-) group in CPH-Ab(+) (P<0.05) and both FCP and PCP were lower than Ab(-) group in GAD-Ab(+) group (P<0.05 and P<0.01). The proportions of patients with insulin deficiency in CPH-Ab(+), GAD-Ab(+), and Ab(-) group were 27.6% (8/29), 48.1% (8/52) and 13.5% (54/400), respectively, which were significantly different among the 3 groups (P<0.001). GAD-Ab, BMI, and fasting blood glucose had effects on FCP and PCP in T2DM patients (All P<0.05), while CPH-Ab did not enter the equation in multivariable stepwise regressive analysis (P>0.05). CONCLUSION: The effect of CPH-Ab is less marked than that of GAD-Ab on islet beta-cell function in LADA patients. The value of CPH-Ab for the failure of islet beta-cell function in LADA should be determined prospectively.

[Etiological dissection in common anti-islet autoantibody-negative patients with type 1 diabetes].

OBJECTIVE: To explore the immunological and genetic factors of common anti-islet autoantibody-negative patients with type 1 diabetes. METHODS: Specimens of peripheral blood were collected from 33 common autoantibody (GAD-Ab, IA2-Ab, IAA, TGA and TPO-Ab) negative diabetic patients with new-onset of unprovoked ketosis (or ketoacidosis), and genome DNA was extracted. The antibodies to carboxypeptide-H (CPH) and SOX13 (ICA12) were detected by radioligand assay. The gene mutations of MODY3 (HNF-1alpha) and MODY6 (NeuroD1/Beta2) were detected by PCR-SSCP sequencing. Mitochondrial gene mutations were analyzed with PCR-RFLP. RESULTS: Two (6%) of the patients were SOX13-Ab positive, while none of them was positive for CPH-Ab. Gene mutation detection found one case of a new mutation, R321H (CGC-->CAC) in the exon 5 of HNF-1alpha gene and one case with ND1 mt3316 G-->A mutation in mitochondrial DNA. In addition to the diabetes-associated mutations described above, seven polymorphisms of HNF-1alpha gene, including L17L, I27L, L459L, S487N, IVS5 + 9 C > G, IVS6-42 G > T, and IVS7 + 7 G > A, and one NeuroD1/Beta2 gene polymorphic variant Ala45Thr, were found. CONCLUSION: Autoimmunity and gene mutations (such as MODY3 and mitochondrial genes mutations) may be etiological in a few cases initially diagnosed as autoantibody-negative type 1 diabetes. Autoimmunity and MODY and mitochondrial diabetes should be excluded if idiopathic type 1 (type 1B) diabetes is diagnosed.

No evidence of pancreatic autoimmunity among patients with multiple sclerosis.

Although multiple sclerosis (MS) usually appears isolated from other autoimmune disorders, an overlap with type 1 diabetes mellitus (T1DM) has been described in Sardinia, where T1DM-associated haplotype HLA-B18-DR3-DQ2 contributes to MS risk. To determine whether in our population MS patients show signs of pancreatic autoimmunity and share this haplotype, sera from 49 MS patients were tested for GAD, IA2, and CPH autoantibodies, and MICA exon 5 polymorphism was genotyped in 30 patients. Pancreatic autoimmune markers were not present among MS patients, nor was any MICA allele associated with MS. Overall, there is no evidence supporting a T1DM/MS overlap in our population.

Diagnostic value of carboxypeptidase-H autoantibodies in detecting latent autoimmune diabetes in adults.

OBJECTIVE: To investigate the diagnostic value of carboxypeptidase-H (CPH) autoantibodies in Chinese patients with the latent autoimmune diabetes in adults (LADA). METHODS: One hundred and fifty-four Type 1 diabetes, 104 Type 2 diabetes, and 144 healthy people were enrolled. Recombinant human CPH (54 kD) was labeled by in vitro translation with 35S-methionine and used to evaluate autoantibodies to CPH (CPH-Ab). Radioimmunoassay was applied to detect antibodies to glutamic acid decarboxylase (GAD-Ab), intracellular part of protein tyrosine phosphatase-like protein (IA2ic-Ab), and autoantibodies to insulin (IAA). RESULTS: No differences in CPH-Ab prevalence were found among Type 1 diabetic patients (5/154, 3.2%), Type 2 diabetic subjects (6/104, 5.7%),and the healthy controls (3/144, 2.1%). The prevalences of GAD-Ab, IA2ic-Ab, and IAA were 15.4% (16/104), 2.9% (3/104), and 2.3% (1/43), respectively in Type 2 diabetes. All IA2ic-Ab or IAA-positive patients with Type 2 diabetes were GAD-Ab-positive. No GAD-Ab- or IAA-positive subjects were observed in CPH-Ab-positive patients with Type 2 diabetes. CONCLUSION: CPH-Ab may provide some diagnostic value for LADA, and improve the sensitivity in diagnosing LADA in Chinese when combined with GAD-Ab test in Type 2 diabetes.

Frequent appearance of autoantibodies against prohormone convertase 1/3 and neuroendocrine protein 7B2 in patients with nonfunctioning pituitary macroadenoma.

Among pituitary disorders having mass effect of the pituitary gland, nonfunctioning pituitary macroadenoma and lymphocytic hypophysitis are difficult to differentiate without histological examination. In order to efficiently distinguish lymphocytic hypophysitis and pituitary tumors, we studied the presence of autoantibodies against prohormone-processing enzymes, prohormone convertase (PC) 1/3, PC2, carboxypeptidase E (CPE), and PC2 regulatory protein, 7B2, by radioligand assay using recombinant human 35S-labeled protein in patients with clinically nonfunctioning pituitary macroadenoma, lymphocytic hypophysitis, and other pituitary diseases. The indexes for anti-PC1/3 antibodies (Ab) were significantly higher in patients with nonfunctioning pituitary macroadenoma than in patients with lymphocytic hypophysitis. Patients positive for either anti-PC1/3 or anti-7B2 Ab were significantly frequent among patients with nonfunctioning pituitary macroadenoma than in other pituitary diseases and healthy controls. None of the patients was positive for anti-PC2 Ab or anti-CPE Ab. These results suggest that autoantibodies against PC1/3 and 7B2 are novel tumor-associated autoantibodies and can be helpful in the diagnosis of clinically nonfunctioning pituitary macroadenoma.