Proteomic analysis of synovial fluid identifies periostin as a biomarker for anterior cruciate ligament injury.

OBJECTIVE: Emerging evidence suggests that injury to the anterior cruciate ligament (ACL) typically initiates biological changes that contribute to the development of osteoarthritis (OA). The molecular biomarkers or mediators of these biological events remain unknown. The goal of this exploratory study was to identify novel synovial fluid biomarkers associated with early biological changes following ACL injury distinct from findings in end-stage OA. METHODS: Synovial fluid was aspirated from patients with acute (≤30 days) and subacute (31-90 days) ACL tears and from patients with advanced OA and probed via tandem mass spectrometry for biomarkers to distinguish OA from ACL injury. Periostin (POSTN) was identified as a potential candidate. Further analyses of POSTN were performed in synovial fluid, OA cartilage, torn ACL remnants, and cultured cells and media by Western blot, PCR, immunostaining and ELISA. RESULTS: Synovial fluid analysis revealed that POSTN exhibited higher expression in subacute ACL injury than OA. POSTN expression was relatively low in cartilage/chondrocytes suggesting it is also produced by other intra-articular tissues. Conversely, high and time-dependent expression of POSTN in ACL tear remnants and isolated cells was consistent with the synovial fluid results. CONCLUSIONS: Elevated POSTN may provide a synovial fluid biomarker of subacute ACL injury setting separate from OA. Increased expression of POSTN in ACL suggests that the injured ACL may play a pivotal role in POSTN production, which is sensitive to time from injury. Previous studies have shown potential catabolic effects of POSTN, raising the possibility that POSTN contributes to the initiation of joint degeneration and may offer a window of opportunity to intervene in the early stages of post-traumatic OA.

Lateralization index but not contralateral suppression at adrenal vein sampling predicts improvement in blood pressure after adrenalectomy for primary aldosteronism.

Adrenal vein sampling (AVS) is essential in differentiating unilateral from bilateral sources of aldosterone excess in primary aldosteronism (PA). However, its ability to predict blood pressure (BP) improvement after adrenalectomy has not been well studied. This is a retrospective observational study of 119 patients who underwent AVS by sequential technique followed by adrenalectomy for PA at the Hospital of the University of Pennsylvania from 1997 to 2015. Median age was 52 years (interquartile range 44-59), 67% were male and median duration of hypertension was 10 (interquartile range 6-20) years. A total of 76% and 90% of patients experienced BP improvement at 0-6 months or at any time point after surgery, respectively. Lateralization index (LI) >8, but not the presence of contralateral suppression, was significantly associated with BP improvement after surgery by multivariate logistic regression analysis adjusted for potential confounders (odds ratio (95% confidence interval): 17.1 (1.7-171.6) and 6.39 (0.06-641.8), respectively). A prediction score was created by covariates that was significantly associated with BP improvement in logistic regression analysis (duration of hypertension, body mass index, preoperative systolic BP and number of antihypertensive medications). Receiver-operating characteristic curve analyses showed that the addition of LI >8 to the score increased its ability to predict BP improvement (area under the curve 0.73-0.80). In conclusion, LI is useful in predicting improvement in BP after adrenalectomy for PA. The results of this study suggest that patients with long-standing severe hypertension may still benefit from surgery if LI >8.

Mortality and cardiovascular disease among older live kidney donors.

Over the past two decades, live kidney donation by older individuals (≥55 years) has become more common. Given the strong associations of older age with cardiovascular disease (CVD), nephrectomy could make older donors vulnerable to death and cardiovascular events. We performed a cohort study among older live kidney donors who were matched to healthy older individuals in the Health and Retirement Study. The primary outcome was mortality ascertained through national death registries. Secondary outcomes ascertained among pairs with Medicare coverage included death or CVD ascertained through Medicare claims data. During the period from 1996 to 2006, there were 5717 older donors in the United States. We matched 3368 donors 1:1 to older healthy nondonors. Among donors and matched pairs, the mean age was 59 years; 41% were male and 7% were black race. In median follow-up of 7.8 years, mortality was not different between donors and matched pairs (p = 0.21). Among donors with Medicare, the combined outcome of death/CVD (p = 0.70) was also not different between donors and nondonors. In summary, carefully selected older kidney donors do not face a higher risk of death or CVD. These findings should be provided to older individuals considering live kidney donation.

Identification of FUSE-binding protein 1 as a regulatory mRNA-binding protein that represses nucleophosmin translation.

Nucleophosmin (NPM/B23) is a multifunctional oncoprotein whose protein expression levels dictate cellular growth and proliferation rates. NPM is translationally responsive to hyperactive mammalian target of rapamycin (mTOR) signals, but the mechanism of this regulation is not understood. Using chimeric translational reporters, we found that the 3' untranslated region (UTR) of the NPM messenger (m)RNA is sufficient to mediate its translational modulation by mTOR signalling. We show that far upstream element (FUSE)-binding protein 1 (FBP1) interacts specifically with the 3' UTR of NPM to repress translation. Overexpression of FBP1 resulted in translational repression of NPM mRNAs, whereas depletion of FBP1 caused a dramatic increase in NPM translation and resulted in enhanced overall cell proliferation. Thus, we propose that FBP1 is a key regulator of cell growth and proliferation through its ability to selectively bind the NPM 3' UTR and repress NPM translation.

Airway proteins involved in bacterial clearance susceptible to cathepsin G proteolysis.

Serine proteases released from neutrophils are central to the pathogenesis of cystic fibrosis lung disease and are considered to be obvious therapeutic targets. Neutrophil elastase digests key opsonins present in the lung and disrupts phagocytosis, allowing bacteria to persist despite established pulmonary inflammation. We have found that cathepsin G, an abundant serine protease found in human and murine neutrophils, has other roles in the development of suppurative lung diseases. Murine models of endobronchial inflammation indicate that cathepsin G inhibits airway defences and interferes with the host's ability to clear Pseudomonas aeruginosa from the lung with effects distinct from neutrophil elastase. We hypothesise that differences in bacterial killing are due to defects in innate defences created by proteolysis. Protein profiles of bronchoalveolar lavage of infected wild-type and cathepsin G-deficient mice were compared using two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry. Four proteins in bronchoalveolar lavage were cleaved by cathepsin G. Serum amyloid P component leaked into the lung during acute infection and was digested by cathepsin G. Its cleavage products had greater binding to lipopolysaccharide and interfered with phagocytosis. These results indicate that cleaved serum amyloid P component acts as an anti-opsonin and interferes with bacterial clearance from the lung.

Change in the pattern of adrenal venous sampling over time in patients with primary aldosteronism.

We report six patients with primary aldosteronism who had serial adrenal venous sampling. Patients with contralateral suppression of aldosterone to cortisol ratio compared with that in inferior vena cava developed lateralization over time whereas patients without contralateral suppression remained with a bilateral pattern.

Correlation between pulse wave velocity and other measures of arterial stiffness in chronic kidney disease.

AIM: Pulse wave velocity (PWV), augmentation index (AIx) and time to first wave reflection (Tr) are all measures of arterial stiffness, but whether these parameters behave similarly in different populations is not well-understood. Given the large burden of cardiovascular disease in individuals with chronic kidney disease (CKD), assessing the relationship between vascular stiffness parameters in this population is important. METHOD: A subset of 152 participants enrolled in the Chronic Renal Insufficiency Cohort Study had vascular stiffness parameters (aortic PWV, central AIx, and Tr) measured using the SphygmoCor system. Linear association between these parameters was assessed using Pearson correlation coefficients. Reproducibility across operators of the device was also tested within individuals. RESULTS: Association was largest between PWV and heart rate-adjusted AIx (AIx-75). The correlation coefficient was 0.371 (p = 0.0003) for ideal studies and 0.305 (p = 0.0001) for all technically acceptable studies. The association between ideal PWV and AIx-75 measurements was 0.361 (p = 0.005) for men and 0.423 (p = 0.01) for women. Bland-Altman plots comparing the mean value of PWV (n = 31) or AIx-75 (n = 21) when measured by 2 different individuals against the difference in their respective values demonstrate that both measures of arterial stiffness are reproducible across multiple technicians. CONCLUSIONS: Thus, we conclude that PWV and AIx-75, despite measuring different quantities in different units, are related measures of arterial stiffness and are reproducible across multiple operators in the population with CKD.

The effects of angiotensin-II on lipolysis in humans.

Adipocytes express many of the proteins of the renin-angiotensin system including angiotensinogen and AT(1)-receptors. A principal function of adipocyte tissue is the provision of energy substrate through lipolysis. This study was undertaken to determine if angiotensin-II (Ang-II) infusion or blockade of the renin-angiotensin system by angiotensin-converting enzyme (ACE) inhibitor therapy with enalapril altered lipolytic activity and substrate oxidation. Eleven healthy male subjects were enrolled in the first study and postabsorptive whole-body lipolysis activity was measured using a stable isotope of glycerol ((2)H(5)-glycerol). Substrate oxidation was determined using indirect calorimetry in the Clinical Research Center. Subjects were then sequentially treated with low-dose Ang-II infusion (0.3 and then 1.0 ng/kg/min) on separate days, and the lipolysis and oxidation studies were repeated. Lastly, each subject was treated with 2 weeks of ACE inhibitor with enalapril (20 mg daily) and underwent lipolysis and oxidation studies for a fourth time. In a second study, 14 healthy male subjects were enrolled and underwent an identical baseline lipolysis and substrate oxidation assessment. These subjects then received an Ang-II infusion at pressor doses (10 ng/kg/min), and changes in lipolytic activity and substrate oxidation were measured again. In the first study, there was no effect on lipolysis activity from low-dose Ang-II infusion (baseline lipolysis activity (mean +/- SD) 2.06 +/- 0.55 micromol/kg/min, 2.10 +/- 0.69 micromol/kg/min after 0.3 ng/kg/min, and 2.32 +/- 0.56 micromol/kg/min after 1.0 ng/kg/min) or enalapril therapy (2.35 +/- 1.00 micromol/kg/min). In the second study, the larger dose of Ang-II increased blood pressure by 14/17 mm Hg, but there was no effect on lipolysis activity (1.36 +/- 0.49 micromol/kg/min v 1.63 +/- 0.82 micromol/kg/min). Substrate oxidation rates were largely unaffected by Ang-II infusions or enalapril therapy. There was no evidence that treatment with subpressor or pressor dosages of Ang-II produced a significant alteration in lipolytic activity. Moreover, blockade of the renin-angiotensin system with enalapril was equally unremarkable in its effects on whole-body lipolysis. These data support the general concept that the renin-angiotensin system in adipocytes serves more to regulate the regional blood flow to adipose tissue and the size and number of fat cells rather than participating directly in the regulation of energy substrate.

Von Hippel-Lindau disease masquerading as autosomal dominant polycystic kidney disease.

The diagnostic confusion in differentiating the various causes of renal cystic diseases in adults is well documented. This confusion can include misclassifications between autosomal dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau disease (VHL). We describe such a case of VHL. A review of the literature and of the patients in our database regarding typical features of each disease, mean age of onset, and frequency of these features was undertaken to provide helpful differentiating features. Pancreatic cysts are one differentiating feature. In VHL, pancreatic cysts can occur in 70% of patients, often are multiple, and rarely may cause exocrine or endocrine insufficiency. Pancreatic islet cell tumors occur. In ADPKD, pancreatic cysts are found in only 9% of patients, usually are single and asymptomatic, generally occur in conjunction with cystic liver disease, and are not found in children or unaffected family members. Pancreatic malignancies do not occur with increased frequency in ADPKD. A different pattern, especially in patients without a strong family history of ADPKD, may be a clue to VHL masquerading as ADPKD. Genetic mutation screening of the VHL gene should be used in these patients.

Dramatic recovery of renal function after 6 months of dialysis dependence following surgical correction of total renal artery occlusion in a solitary functioning kidney.

Revascularization of renal artery stenosis for the treatment of hypertension is an established procedure. In selected clinical scenarios, successful revascularization procedures may preserve or restore renal function. We present a 31-year-old man who underwent successful renal revascularization of a solitary functioning kidney after being dialysis dependent for approximately 190 days. He had dramatic improvement of renal function and has remained off dialysis since his surgery 18 months ago. He continues to have severe but controllable hypertension.

Safety of intravenous gadolinium (Gd-BOPTA) infusion in patients with renal insufficiency.

The safety of gadolinium (Gd-benzyloxypropionictetra-acetate [BOPTA] dimeglumine) infusion was evaluated in 32 patients with severe or moderate chronic renal failure in a prospective, randomized, double-blind, placebo-controlled study. Renal failure was defined as severe if creatinine clearance was between 10 and 29 mL/min, and as moderate if creatinine clearance was between 30 and 60 mL/min. Serum creatinine level and 24-hour urine samples for creatinine clearance were followed up serially for 7 days after the administration of either gadolinium (Gd-BOPTA dimeglumine), 0.2 mmol/kg, or a saline infusion. No patient experienced a significant change in renal function, defined as an increase in serum creatinine level greater than 0.5 mg/dL more than baseline, and no patient required hospitalization or dialysis during the study period. Gadolinium (Gd-BOPTA dimeglumine) appears to be well tolerated in patients with moderate to severe renal failure.

Glycan composition of serum alpha-fetoprotein in patients with hepatocellular carcinoma and non-seminomatous germ cell tumour.

Although estimation of serum alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumours (NSGCT), the clinical usefulness of this test is limited by a low specificity. However, there exist glycoforms of AFP which may be more specific for particular tumours. Previously, detailed analysis has been prevented by the low levels of AFP in human serum. We report here the application of fluorescence labelling, sequential exoglycosidase digestion, high-performance liquid chromatography and matrix-assisted laser desorption ionization in time-of-flight mass spectrometry, to determine the glycan structures of purified serum AFP from patients with HCC and NSGCT. Eleven major glycans were found, of which seven were N-linked, and four were O-linked, to the protein backbone. The structure of the N-linked glycans (all of bi-antennary complex-type with varying degrees of sialylation, fucosylation and galactosylation) were consistent with those previously reported. The O-linked glycans (three mucin O-GalNAc type glycans with variable degrees of sialylation, one O-HexNAc monosaccharide glycan) have not previously been reported. The finding of mucin O-GalNAc type glycans was supported by the prediction of potential O-GalNAc glycosylation sites on the protein backbone by analysis of the AFP structure by molecular modelling. With knowledge of these structures it may be possible to develop more specific assays for the detection of HCC and NSGCT.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.