Nuclear matrix protein is released from apoptotic white cells during cold (1-6 degrees C) storage of concentrated red cell units and might induce antibody response in multiply transfused patients

BACKGROUND: A previous study showed that white cells in blood units undergo apoptosis during storage. STUDY DESIGN AND METHODS: The present study attempts to show the release of nuclear matrix protein (NMP) in the supernatants of red cell units and to determine whether antibodies against nuclear components may be present in multiply transfused patients; the methods employed were enzyme-linked immunosorbent assay, flow cytometry, microscopy, immunoblotting, immunofluorescence, and confocal laser-scanning microscopy. RESULTS: NMP is released from white cells in the supernatant of packed red cell units upon cold storage (1-6 degrees C). The concentration of NMP correlates well with the degree of apoptosis, as analyzed by flow cytometry, nuclear dye staining, and DNA gel electrophoresis. Immunofluorescence also shows that white cells undergoing apoptosis (pre-G(1) peak, as seen by propidium iodide staining and flow cytometry) have an NMP content lower than control cells, which confirms an actual release of NMP. Moreover, immunoblotting analysis and immunofluorescent staining showed that, in 4 of 38 multiply transfused patients, autoantibodies against NMPs were present without any clinical or laboratory sign of autoimmune disease. One of the sera, recognizing a 64-kDa NMP, immunostained nuclear dots that were identified as coiled bodies because of their colocalization with p 80 coilin. CONCLUSION: NMP is released in the supernatant of red cell units. The results obtained from patients suggest that nuclear proteins released during apoptosis, once transfused, may induce an immune response in multiply transfused patients.     

Defining the role of NMP22 in bladder cancer surveillance

Despite advances in treatment and knowledge of its pathogenesis, urothelial carcinoma of the bladder remains a significant cause of morbidity and mortality. Experience with the natural course of bladder cancer has revealed that early diagnosis of primary and recurrent disease improves patient prognosis. In this regard, cystoscopy (usually in combination with urinary cytology) has long been regarded as the gold standard for the diagnosis and surveillance of bladder cancer. However, the disadvantages inherent to cystoscopy, including invasiveness and cost, have stimulated a search for alternative methods for detecting urothelial malignancy. The ideal alternative test would duplicate the high accuracy of cystoscopy for detecting bladder tumors while eschewing its invasiveness, attendant morbidity, and high cost. The vast majority of bladder cancers arise from the urothelium, which continually sheds cells as well as intracellular contents into the urine, thereby providing a potential source of cancer-specific markers. Voided cytology and urinalysis are established tests that have been the standard tools for detection of such substances. The last decade has seen the rise of a myriad of novel urine-based bladder tumor markers, including bladder tumor antigen, urinary bladder cancer antigen, fibronectin, telomerase, and nuclear matrix proteins (e.g., NMP22). The NMP22 assay in particular has been the subject of considerable study and has demonstrated some promise as a potential adjunct to cystoscopy and cytology. Through a critical review of the literature, we seek to define the role, if any, of NMP22 in the follow-up of patients with a previous history of urothelial carcinoma of the bladder.     

Bladder tumor markers: Need, nature and application. 1 Nucleus-based markers

Urothelial tumors are common: their diagnosis and long-term management represent a large part of most urologists' workload. The majority of such tumors are superficial and are mostly managed by repeated cystoscopic surveillance and treatment. A smaller but significant group of patients either start with, or subsequently progress to, more invasive disease, thus requiring an alternative and more invasive treatment. Maximizing the benefit/risk ratio of the diagnosis and the various treatment options of bladder tumors requires the availability of a reliable tumor marker. The concept of tumor markers encompasses the utilization of any detectable deviation from normality that is indicative of neoplasia. For bladder cancer, most of these markers are present in urine. In this part of the review we examine, from the clinician's point of view, the literature verdict on older techniques such as cytology and cytometry, as well as the current status of new nucleus-based tests such as P53, telomerase, NMP22 and Ki67.     

Solvent effects on coupling yields during rapid solid‐phase synthesis of CGRP(8‐37) employing in situ neutralization*

Abstract: The success of solid‐phase peptide synthesis is often dependent upon solvation of the resin and the growing resin‐bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide‐resin in order to obtain satisfactory coupling yields for the rapid solid‐phase peptide synthesis, using butyloxycarbonyl‐(Boc)‐amino acid derivatives, of human‐α‐calcitonin gene‐related peptide(8‐37) (CGRP(8‐37)). Solvation of (p‐methylbenzhydrylamine)copoly(styrene–1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8‐37) (peptide–resin) was correlated to solvent Hildebrand solubility (δ) and hydrogen‐bonding (δ_h) parameters. Contour solvation plots of δ_h vs. δ revealed maximum solvation regions of resin and peptide–resin. Maximum resin solvation occurred with N‐methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide–resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide–resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide–resin solvation shifts to solvents with higher δ_h values. DMF provided the most effective peptide–resin solvation and was the only solvent from which CGRP(8‐37) was obtained as a single major product in the crude cleaved material.     

尿端粒酶、NMP22和ImmunoCyt联合检测在膀胱癌诊断中的应用

目的评价尿端粒酶、核基质蛋白22（NMP22）和ImmunoCyt测定在膀胱癌（BTCC）早期诊断中的应用价值,寻找早期诊断膀胱癌的有效方法。方法选择60例经病理诊断明确为早期膀胱移行细胞癌和60例非膀胱肿瘤患者,分别进行尿端粒酶、ImmunoCyt、NMP22和标准尿脱落细胞学检测。结果尿端粒酶、NMP22和ImmunoCyt和尿脱落细胞学的敏感度分别为58.3%、60.0%、61.6%、26.6%;准确度分别为75.8%,78.3%,78.3%和63.3%;特异度分别为93.3%,96.6%,95.0%和100%。三者联合检测与尿端粒酶、NMP22和ImmunoCyt单独检测相比,敏感度（分别为χ2=6.53,P〈0.05;χ2=5.17,P〈0.05;χ2=4.54,P〈0.05）和准确度（分别为χ2=4.01,P〈0.05,χ2=4.38,P〈0.05,χ2=4.91,P〈0.05）都有所提高;尿端粒酶、NMP22和ImmunoCyt联合检测的敏感度（χ2=28.06,P〈0.01）和准确度（χ2=16.2,P〈0.01）均高于尿脱落细胞学,差异有统计学意义。结论尿端粒酶、NMP22和ImmunoCyt是早期诊断膀胱移行细胞癌的一种高特异性指标,三者联合检测能提高膀胱癌的早期诊断率。     

Evaluation of NMP179 for the detection of squamous intraepithelial neoplasia in ThinPrep cervical slides using a combination of double immunostaining and morphometric methods of analysis

This study investigates the potential value of the nuclear matrix protein NMP179 as a marker of abnormal squamous cells in ThinPrep slides. Forty-six cervical scrapes were collected as cell suspensions and ThinPrep slides were prepared. They were double-immunostained for NMP179 and Cytokeratin 18 (CK18), an endocervical cell marker. The method of analysis adopted for the study was designed to distinguish the abnormal squamous cells from benign epithelial cell so that the percentages of abnormal squamous cells that expressed the marker could accurately be determined. Initially, an attempt was made to identify benign and abnormal cells in the ThinPrep slides on the basis of their morphology and immunostaining patterns. Discrimination between the various types of epithelial cells was incomplete using this approach and a more precise method of discrimination between the different epithelial cell types was carried out using a combination of double immunostaining (NMP179 and CK18) and morphometry using nuclear area and nuclear cytoplasmic ratios. Once the different epithelial cell types had been identified, the specificity and sensitivity of NMP179 were determined. The optimal sensitivity (89.9%) was achieved at the N/C ratio 0.36; however, the specificity of NMP179 was very low for all N/C ratios and ranged from 38.8% to 42.2%.     

The role of haematuria in bladder cancer screening among men with former occupational exposure to aromatic amines

Study Type – Diagnostic (validating cohort) Level of Evidence 1b What's known on the subject? and What does the study add? Microscopic haematuria (µH) is frequently detected in elderly adults. The American Urological Association recommends the follow‐up of subjects with µH on bladder cancer. Whereas gross haematuria is considered an important sign of the presence of bladder cancer, the disease‐predictive value of µH is less clear. No association of µH with the development of bladder tumours in a prospective screening cohort of chemical workers was observed. The positive predictive value of µH for bladder cancer was as low as 1.2%. Haematuria interfered with NMP22 but not with cytology and UroVysion^TM test results.

OBJECTIVE

? To assess the positive predictive value (PPV) of microhaematuria (µH) and gross haematuria (GH) in bladder cancer screening and the influence of haematuria on tumour tests in a prospective study.

PATIENTS AND METHODS

? From September 2003 to January 2010, 1323 men took part in an annual voluntary bladder cancer screening programme for chemical workers with former exposure to aromatic amines. ? In 5315 urine samples haematuria was determined with a dipstick, followed by a microscopic blood cell count in the sediment. Haematuria was categorized into traces, µH and GH. ? Urinary leukocytes and other factors were investigated as potential predictors of haematuria using a generalized estimating equation model for repeated urinalysis. The risk of haematuria for positive tumour tests was analysed correspondingly. ? The bladder cancer risk was estimated for the highest degree of haematuria occurring during the study with Poisson regression.

RESULTS

? As of July 2010, 15 bladder tumours were detected in 14 participants. ? GH was found in four out of nine high‐grade tumours and associated with a rate ratio of 3.82, 95% confidence interval (CI) 0.50–29.15 for the development of bladder lesions. ? The PPV of GH was 11.4%, but only 1.2% for µH. µH occurred in 18.8% of urine samples and was not associated with bladder cancer [rate ratio (RR) 0.72, 95% CI 0.11–4.78]. ? Abundant urinary leukocytes were associated with µH [odds ratio (OR) 8.34, 95% CI 2.26–30.69] and even stronger with GH (OR 22.25, 95% CI 6.42–77.06). ? Haematuria and leukocytes influenced NMP22 positivity (µH: OR 1.63, 95% CI 1.06–2.51, abundant leukocytes: OR 8.90, 95% CI 1.58–50.16), but not test results for urine cytology and UroVysion^TM.

CONCLUSION

? While the PPV of µH for bladder cancer was low, there was a strong influence of haematuria and leukocytes on the protein‐based tumour test NMP22®. ? Erythrocytes and leukocytes should be determined at least semi‐quantitatively for the interpretation of positive NMP22 test results. ? In addition, a panel of tumour tests that includes methods not affected by the presence of erythrocytes or leukocytes such as cytology and UroVysion^TM would improve bladder cancer screening.