Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples.

We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we "count" the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity-based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester-hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.

Low-molecular-weight Protein Tyrosine Phosphatase Is a Possible Biomarker for Predicting Postoperative Biochemical Recurrence in Prostate Cancer With Negative Surgical Margins.

BACKGROUND/AIM: For prostate cancer, positive surgical margins are considered an important predictor of biochemical recurrence. However, biochemical recurrence is observed in approximately 20% of cases, even with negative surgical margins, and some cases require salvage therapy. The elevated expression of low-molecular-weight protein tyrosine phosphatase (LMW-PTP, MW 18 kDa) is associated with a poor prognosis of certain cancers. In this study, we investigated whether the LMW-PTP expression levels could be used as a biomarker of recurrence in prostate cancer with negative surgical margins. MATERIALS AND METHODS: The subjects of this retrospective study were 119 patients who underwent total prostatectomy with negative resection margins. LMW-PTP expression was categorized either as a high-expression group or as a low-expression group bye two pathologists. Subsequently, we examined the relationship between LMW-PTP expression levels and clinicopathological factors including biochemical recurrence. RESULTS: Evaluation of the immunostained samples by two pathologists was highly reliable, with an Intraclass correlation (ICC) score for two distinct measurements of 0.77 and 0.98, respectively. Seventy-three patients (61.3%) were placed in the LMW-PTP high expression group; and 46 patients (38.7%) were placed in the low expression group. The log-rank test revealed early biochemical recurrence in the high LMW-PTP expression group (p=0.0001). In addition, pathological T stage (p=0.004), lymphatic invasion (p=0.0456), Ki-67 labeling index (p=0.0002), and biochemical recurrence (p<0.0001) were more frequently identified in the LMW-PTP high expression group. Furthermore, multivariate analyses revealed that a high LMW-PTP expression level was an independent prognostic factor for biochemical recurrence (HR=3.14, 95% CI=1.37-8.07, p=0.0057). In addition, Ki-67 labeling indices were significantly higher in the high-expression group compared to the low-expression group (p<0.0001). CONCLUSION: LMW-PTP can be assessed using a single immunostaining protocol in a highly reproducible fashion. Tt may, thus, be applied clinically to establish the required postoperative follow-up period and determine the necessity for salvage therapy in cases of prostate cancer with negative surgical margins. LMW-PTP has the potential to be a highly useful prognostic biomarker and a therapeutic target in conjunction with other factors, such as the Gleason Score, the pathological T stage and the PSA level.

High levels of antibodies against PtpA and PknG secreted by Mycobacterium avium ssp. paratuberculosis are present in neuromyelitis optica spectrum disorder and multiple sclerosis patients.

Mycobacterium avium ssp. paratuberculosis (Map) is the etiological agent of Paratuberculosis in ruminants. Protein tyrosine phosphatase A (PtpA) and protein kinase G (PknG) are secreted proteins necessary for the survival of the pathogen within macrophages. In this study we analyzed if Map was able to grow within astrocytes and investigated on the presence of antibodies against PtpA and PknG proteins in MS and NMOSD patients by ELISA. Map was unable to proliferate in astrocytes after of 72 h post-infection, but we observed a high level of antibodies against both virulence factors, suggesting that these patients have been exposed/infected with Map.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance

ABSTRACT Objective We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. Subjects and methods Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. Results There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. Conclusion A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance.

OBJECTIVE: We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. SUBJECTS AND METHODS: Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. RESULTS: There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. CONCLUSION: A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

From bench to bedside: bipolar androgen therapy in a pilot clinical study.

Prostate cancer remains a leading cause of cancer death in Europe and the United States and is an emerging problem in Asia despite significant improvements in available treatments over the last few decades. Androgen deprivation therapy (ADT) has been the core treatment of advance-staged disease since the discovery of prostate cancer's androgen dependence in 1941 by Huggins et al. [1] Options for initial medical treatment include gonadotropin-releasing hormone analogues such as leuprolide (LHRH agonist) and degarelix (LHRH antagonist) and androgen receptor (AR) binding agents such as bicalutamide. Although most patients will initially respond to either surgical or medical castration, there is almost always progression to castration-resistant prostate cancer (CRPC) necessitating treatment with more novel agents. [2] However, even drugs such as abiraterone and enzalutamide, two next-generation agents used commonly in metastatic CRPC, have failed to demonstrate persistent efficacy in most patients. [3] ,[4].

Effects of E/Z isomers of lycopene on experimental prostatic hyperplasia in mice.

AIM: Lycopene is a member of the carotenoid family and has strong anti-oxidant properties. Lycopene occurs in tomato-based food products primarily as an all-E isomer (80-97%),but its Z-isomers accounts for 79 to 88% of total lycopene in benign or malignant prostate tissues, while the specific biological functions of Z-isomers are still not clarified at present. This study was to examine the bioactive potency of Z-isomers on benign prostatic hyperplasia (BPH) in mice and to make a comparison of effective inhibition between Z-isomers and all-E isomer. METHOD: Mice were divided into the Saline group, Vehicle control group and testosterone propionate induced BPH mice group (BPH model group, vehicle BPH model group, lycopene treated (5 mg/kg and 2.5 mg/kg), Z-isomers (57%) treated, Z-isomers (86%) treated, finasteride treated). The drugs were orally administered once a day consecutively for 30 days. The inhibitory effects on BPH of all-E lycopene and Z-isomers were evaluated by prostatic index, prostatic acid phosphatase (PAP), estradiol, testosterone and dihydrotestosterone (DHT) levels in serum and histopathology examination. RESULTS: Compared with the BPH model group, E/Z isomers exhibited significant differences in prostatic index, PAP, estradiol, testosterone and DHT levels in serum and similar histological aspects observed in the mice of the control group. The present research also shows that Z-isomers may be more potent inhibitors than all-E isomers in BPH treatment.

An unusual variant of prostatic adenocarcinoma with metastasis to testis. A case report.

Ductal adenocarcinoma of the prostate is considered to be a rare variant of prostatic adenocarcinoma when compared to the more common acinar adenocarcinoma. We report here a case of ductal adenocarcinoma of the prostate in a 68-year old man who presented with complaints of abdominal pain, retention of urine and hematuria of one month duration. Clinical examination showed prostatomegaly. The serum Prostate Specific Antigen (PSA) value was raised to 79ng/mL. Histopathological and immunohistochemical evaluation of resected specimen of prostate revealed ductal adenocarcinoma of the prostate. The patient was lost to follow up and presented four years after the initial diagnosis with metastasis to the bone and testis. Though prostatic cancers have the ability for wide spread dissemination, metastasis to testis is rare. Immunohistochemical staining with PSA and Prostatic Acid Phosphatase (PAP) can help in establishing prostatic nature of the neoplasm. We are reporting this case because of the rarity of metastasis of prostatic carcinoma to testis and for stressing the need for keeping in mind the possibility of metastatic carcinoma also while dealing with testicular tumors.

Erythrocyte deformability dependence on band 3 protein in an in-vitro model of hyperfibrinogenemia.

Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the erythrocyte deformability was determined. The present study shows that in the presence of higher fibrinogen concentrations, similar to those found in inflammatory conditions, erythrocyte deformability is increased only when band 3 is dephosphorylated by the presence of syk inhibitor and at low shear stress. On the contrary, no changes were verified in the presence of fibrinogen when band 3 is allowed to be phosphorylated by inhibiting the phosphotyrosine phosphatase enzyme activity with calpeptin. We also observed that the presence of fibrinogen at higher concentration does not induce changes in erythrocyte deformability in the absence of modulators of the band 3 phosphorylation degree. However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study.

Inhibition of the experimental induction of benign prostatic hyperplasia: a possible role for fluted pumpkin (Telfairia occidentalis Hook f.) seeds.

INTRODUCTION: Pumpkins are thought to be useful in the management of benign prostatic hyperplasia (BPH). The ability of a 15% Telfairia occidentalis seeds incorporated diet to inhibit hormonal induction of BPH in rats was studied. MATERIALS AND METHODS: Twenty male Wistar rats were divided into 4 equal groups - one test group and three control groups. The test group was placed on the test diet and was given subcutaneous injections of dihydrotestosterone (DHT) and estradiol valerate (ratio 10:1) every other day for 28 days. One control group, 'no test diet' (ND) group, received the hormones, but was placed on a normal diet. The other two control groups, 'no hormone' (NH) and 'no hormone/test diet' (NHD), received subcutaneous olive oil (vehicle) for the same duration and were placed on the test and normal diets, respectively. Markers of BPH and hormone profile were determined using standard methods. RESULTS: The mean relative prostate weight (×10(3)) was reduced in the test group (3.6 ± 0.2) relative to the ND group (4.0 ± 0.4). The protein content (mg/tissue) of the rats' prostates decreased significantly (p < 0.05) from 68.3 ± 2.7 in the ND group to 43.4 ± 3.9 in the test group. Serum prostatic acid phosphatase levels (U/l) decreased significantly (p < 0.05) from 4.8 ± 0.4 in the ND group to 4.0 ± 0.9 in the test group. Histological findings corroborate these data. The testosterone:estradiol ratio (×10(3)) was significantly (p < 0.05) increased from 7.1 ± 0.1 in the ND group to 8.4 ± 0.4 in the test group. CONCLUSION: The test diet inhibited the induction of BPH in rats and may act by increasing the testosterone:estradiol ratio.

[Alternative tests to PSA for prostate cancer diagnosis]. / Nuovi test in competizione con il PSA per la diagnosidel carcinoma prostatico.

Prostate specific antigen (PSA) is still the most useful tool to select the population requiring prostatebiopsy. The main downsides of PSA are an inadequate sensitivity to be used in screening and a low specificity for cancer detection. So far, a limited value for PSA derivates (velocity, density, free, proisoforms and doubling time) has been recognised. We present a short review of the literature describing a selection of the most promising alternatives to PSA being studied currently: PCA3, serum kallikreins, serum detectable prostate specific membrane antigen, the nuclear matrix protein EPCA, EPCA-2, prostatic acid phosphatase, urine detectable GSTP1, anti-AMACR antibodies, sarcosine, plasminogen activating urokinase, IGFBP, TGF beta 1,PSP94, IL6, plasmatic DNA, serum autoantibodies, neuroendocrine markers, proteomic analysis.

Adenoid cystic carcinoma of the prostate: case report on a rare entity and review of the literature.

Adenoid cystic carcinoma is an unusual histological variant of prostatic carcinoma. Because of its rarity, the natural history of this tumor is not known. Here we report this rare entity in a 62-year-old man who presented with symptoms of urinary tract obstruction. Digital rectal examination and ultrasonography (USG) showed an enlarged hard nodular prostate. Serum prostate-specific antigen (PSA) and prostatic acid phosphate levels were found to be within the normal range. Transrectal ultrasound-guided 12 core biopsies of prostate showed morphological features of an adenoid cystic carcinoma in 8 cores (bilateral, mid and base) on histopathological examination. Immunohistochemistry performed for PSA on paraffin section was negative. After diagnosis, bilateral orchidectomy was performed, and hormonal therapy was started in the form of androgen receptor blocker. The patient was clinically stable during a limited follow up of six months.

Fulminant type 1 diabetes mellitus exhibits distinct clinical and autoimmunity features from classical type 1 diabetes mellitus in Chinese.

BACKGROUND: fulminant type 1 diabetes mellitus (T1DM) is characterized by abrupt onset of hyperglycemia and rapid progression to ketoacidosis. This study aimed at examining the clinical and autoimmunity features of fulminant T1DM in Chinese. METHODS: a total of 24 patients with fulminant T1DM and 48 classical autoimmune T1DM patients with acute onset of ketoacidosis were recruited. Anthropometric and biochemical parameters were tested. Serum antibodies against glutamic acid decarboxylase 65, tyrosine phosphatase-2 and zinc transporter 8 were measured, and human leukocyte antigen-DQ haplotypes were determined. Peripheral glutamic acid decarboxylase 65-specific T-cell responses in some subjects were determined. RESULTS: compared with autoimmune T1DM patients, patients with fulminant T1DM displayed more flu-like and gastrointestinal symptoms, and had significantly higher concentrations of plasma glucose, more severe ketoacidosis, very low levels of pancreatic ß-cell reserve, but only slightly increased haemoglobin A(1c) levels. In some patients, the disease onset was associated with drug-related hypersensitivity, pregnancy, or positive serum IgM against viruses. Forty percent (8/20) had low titres of autoantibodies against at least one of the islet autoantigens tested. Three out of six patients had positive glutamic acid decarboxylase-reactive Th1 responses. The frequency of human leukocyte antigen -DQA1*0102-DQB1*0601 haplotype was significantly higher in patients with fulminant T1DM. CONCLUSIONS: fulminant T1DM is a distinct entity with unique clinical characteristics and may be mediated by multiple factors, including viral infection, pregnancy, and drug sensitivity syndrome, among others, in the presence of humoral and/or cellular autoimmunity and susceptible genetic background.

PTPIP51-a myeloid lineage specific protein interacts with PTP1B in neutrophil granulocytes.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) was identified as an in vitro interacting partner of protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP). The full-length form of PTPIP51 encompasses 470aas and has a molecular weight of 52kDa. The physiological function is poorly understood but an involvement in differentiation processes and apoptosis has been suggested. Preliminary observations suggested differences in PTPIP51 expression in blood cells. To analyze a possible involvement of PTPIP51 in hematopoietic processes, we studied its expression in samples of peripheral venous blood (PVB), umbilical cord blood (UCB) and human bone marrow (HBM). In both, PVB and UCB PTPIP51 expression was restricted to neutrophil granulocytes. In HBM samples, besides in mature neutrophil ganulocytes PTPIP51 protein and mRNA was present in myeloid precursor cells of neutrophils. The expression of PTPIP51 in neutrophil granulocytes was corroborated by immunoblot analysis exhibiting different molecular weight forms of PTPIP51 protein. Anti-peptide antibodies, identifying specific regions of the PTPIP51 protein (C-terminus, N-terminus and aas114-129) revealed a distinct isoform expression pattern in neutrophil granulocytes of different sources. In PVB and UCB neutrophil granulocytes reacted positive for all three peptide antibodies. In contrast, neutrophils of HBM express solely an N-terminal variant of PTPIP51 protein, lacking the C-terminal and aas114-129 sequence. Immunocytochemical results displayed a strict co-localization of PTPIP51 and PTP1B in PVB and UCB. The interaction of both proteins was verified by a proximity ligation assay. Neither proliferating cells, as identified by PCNA immunostaining, nor apoptotic cells, labeled by TUNEL assay, displayed an immunoreactivity for PTPIP51 in HBM. In fact, PTPIP51 expression was restricted to myeloid precursor cells undergoing differentiation. In blood cells therefore, PTPIP51 expression is restricted to differentiating and mature neutrophil granulocytes.

Glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids.

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are glycoproteins secreted by prostate epithelial cells, and have a long clinical history of use as serum biomarkers of prostate cancers. These two proteins are present at significantly higher concentrations in seminal plasma, making this proximal fluid of the prostate a good source for purifying enough protein for characterization of prostate disease associated changes in glycan structures. With the use of seminal fluid samples representative of normal control, benign prostatic disease and prostate cancers, PAP and PSA were enriched by thiophilic absorption chromatography. Released N-linked glycan constituents from both proteins were analyzed by a combination of normal phase HPLC and MALDI-TOF spectrometry. For PSA, 40 putative glycoforms were determined, and 21 glycoforms were determined for PAP. PAP glycans were further analyzed with a hybrid triple quadrupole/linear ion trap mass spectrometer to assign specific glycoform classes to each of the three N-linked sites. The glycans identified in these studies will allow for more defined targeting of prostate disease-specific changes for PAP, PSA and other secreted prostatic glycoproteins.

The effects of ammonium metavanadate on biochemical, hormonal, haematological and histopathological parameters of the female Wistar rats.

The effects of different doses of ammonium metavanadate on the biochemical, haematological, hormonal and histopathological parameters of stilbesterol treated female Wistar rats were investigated. Ammonium metavanadate in the dose range 0-6 mg/kg caused a bi-phasic and time-dependent response on the acid [total and prostate] phosphatase. Furthermore ammonium metavanadate caused a dose-dependent inhibition of the serum alkaline phosphatases. The maximal inhibitory response at 5mg/kg of ammonium metavanadate was 40.0+/-1.69 compared to 65.0+/-0.94 control values. Ammonium metavanadate also caused a positively correlated biphasic response in the serum female hormonal concentrations with an initial increase, followed by a time-dependent decrease in the serum values of luteinizing (LH), follicle stimulating hormone (FSH) and prolactin. Furthermore ammonium metavanadate also caused time-and dose-dependent effects on the haematological parameters. The effects were biphasic-increase within 72 hours and a reduction in the values of haemoglobin and packed cell volume within 7-28 days. The white blood count and lymphocyte counts were also reduced [P<0.05] significantly. However the neutrophil counts were increased dose-and time-dependently. Finally, ammonium metavanadate caused a dose-dependent destruction of the liver and female reproductive organs namely the uterus, ovary and fallopian tubes. These were characterized by necrosis, oedema, eosinophilic deposits and vacuolation. These results may be explained by the oxidative effects caused by the free oxygen [O2] radical generated by the metavanadate ions.

Antioxidants, cadmium-induced toxicity, serum biochemical and the histological abnormalities of the kidney and testes of the male Wistar rats.

The effect of different doses of cadmium [CD] on some biochemical, hormonal and histopathological parameters of the liver, kidney and testes of the Wistar rate were investigated. Cadmium in the dose range 0-40 mg/kg while causing a time-and dose-dependent decrease of the basal serum levels of alkaline phosphatase [ALP] also caused a dose-dependent increase in the serum concentration of the acid and prostatic acid phosphatases. The value of the ALP changed from 148.7+/-1.0 IU/L in the control to 53.7+/-0.098 at 40 mg/kg of cadmium. While the ACP and ACPT changed from 32.6+/-0.72 and 7 Units in the control to 54 and 17 units respectively at 40 mg/kg of CD. Furthermore cadmium also caused positively correlated dose-and time-dependent destruction of the histology of the liver, kidney and testes. These were characterized by vascular congestion, vacuolation, destruction of the seminal epithelial layers, focal necrosis of nucleus, oedema of the seminal epithelia layers, focal necrosis of nucleus, oedema of the seminiferous tubules and reduction of spermatogenesis. CD also caused granular and eosinophilic cytoplasm, enlargement of sinusoids with kupffer cells, haemorrhage and apoptosis of cells. Finally pre-treatment with vitamin C [0.0015/kg], vitamin E [1.51/g] and selenium [0.25 mg] which on their own had little or no effects on the serum basal phosphatases, hormonal and histological stability caused a reversal of the cadmium-induced biochemical, hormonal and histological toxicities of the liver, kidney and testes. These results may be explained by the oxidational/antioxidational properties of these xenobiotics and their mechanisms of actions.

Prostatic acid phosphatase adversely affects cause-specific survival in patients with intermediate to high-risk prostate cancer treated with brachytherapy.

OBJECTIVES: To perform a retrospective analysis to assess the utility of pretreatment serum prostatic acid phosphatase (PAP) as a predictor of cause-specific survival (CSS) in patients with higher risk prostate cancer treated with palladium-103 (103Pd) brachytherapy and supplemental external beam radiotherapy (EBRT). METHODS: From 1992 to 1996, 193 patients with clinically localized prostate adenocarcinoma, a pretreatment PAP level, and Gleason score 7 or more, and/or a prostate-specific antigen (PSA) level of 10 ng/mL or more were treated with 103Pd brachytherapy and supplemental EBRT. The patients underwent EBRT of 41.4 Gy to a limited pelvic field and 103Pd brachytherapy with a prescribed minimum 103Pd dose of 80 Gy. Multivariate analysis was performed to analyze the predictive value of PAP, PSA, and Gleason score on CSS. RESULTS: The 10-year CSS rate for patients with a PAP level of less than 1.5, 1.5 to 2.4, and 2.5 U/L or more was 93%, 87%, and 75%, respectively (P = 0.013). The 10-year CSS rate for patients with a PSA level of less than 10, 10 to 20, and greater than 20 ng/mL was 92%, 76%, and 83%, respectively (P = 0.393). The 10-year CSS rate for patients with a Gleason score of 6, 7, 8, and 9 was 90%, 89%, 70%, and 68%, respectively (P = 0.002). On Cox multivariate regression analysis, PAP (hazard ratio 1.31, P <0.0001) and Gleason score (hazard ratio 2.37, P = 0.0007) were associated with CSS. PSA was not predictive of CSS (P = 0.393). CONCLUSIONS: The results of this study demonstrated that PAP is a stronger predictor of CSS than PSA or Gleason score in men with higher risk prostate cancer treated with 103Pd brachytherapy and EBRT. Given the findings of this analysis, the use of PAP should be reconsidered in these patients.