Certification of creatinine in a human serum reference material by GC-MS and LC-MS

BACKGROUND: To meet recommendations given by the Laboratory Working Group of the National Kidney Disease Education Program for improving serum creatinine measurements, NIST developed standard reference material (SRM) 967 Creatinine in Frozen Human Serum. SRM 967 is intended for use by laboratories and in vitro diagnostic equipment manufacturers for the calibration and evaluation of routine clinical methods. METHODS: The SRM was produced from 2 serum pools with different creatinine concentrations. The concentrations were certified using a higher-order isotope-dilution GC-MS method and an isotope-dilution LC-MS method. The LC-MS method is a potential higher-order reference measurement procedure. RESULTS: The GC-MS mean (CV) concentrations were 67.0 (0.9%) mumol/L for serum pool 1 and 346.1 (0.45%) mumol/L for serum pool 2. The LC-MS results were 66.1 (0.2%) mumol/L and 346.3 (0.2%) mumol/L, respectively. For serum pool 1, there was a 1.4% difference between the mean GC-MS and LC-MS measurements, and a 0.10% difference for serum pool 2. The results from the 2 methods were combined to give the certified concentrations and expanded uncertainties. CONCLUSIONS: The certified concentration (expanded uncertainty) of SRM 967 was 66.5 (1.8) mumol/L for serum pool 1 (a value close to the diagnostically important concentration of 88.4 mumol/L) and 346.2 (7.4) mumol/L for serum pool 2 (a concentration corresponding to that expected in a patient with chronic kidney disease).     

Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.     

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil

BackgroundBaloxavir marboxil (BXM) is an approved drug that selectively targets cap‐dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10‐fold.MethodsWe comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity.ResultsPA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment‐emergent substitutions in the CAPSTONE‐2 study. The I38N substitution conferred reduced susceptibility by 24‐fold, whereas replicative capacity of the I38N‐substituted virus was impaired compared with the wild‐type. The I38R‐substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4‐fold change).ConclusionThese results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.     

Ciliated muconodular papillary tumor of the lung: A newly defined peripheral pulmonary tumor with conspicuous mucin pool mimicking colloid adenocarcinoma: A case report and review of literature

We report the case of a 68‐year‐old man with a newly defined rare entity of a peripheral pulmonary tumor, consisting of a nodular papillary lesion with papillary structures containing ciliated columnar and goblet cells, as well as floating tumor cells in the mucin pool. The conspicuous mucin pool was observed to be mimicking colloid adenocarcinoma in a low‐power view, particularly in a frozen section slide. We originally reported it as an adenocarcinoma during intraoperative consultation. Immunohistochemically, the tumor cells exhibited a similar immunophenotype to pulmonary adenocarcinoma, except for the presence of focal ciliated and basaloid cells, which we found using CK5/6 and P63 immunostaining. No KRAS or EGFR mutation was found. We revised the diagnosis to that of a ciliated muconodular papillary tumor (CMPT). Four years after a wedge resection, the patient remained free of tumors. Although the malignant potential of CMPT cannot be ignored, a wedge resection with a safe margin might be a treatment option for CMPT patients.     

The topoisomerase II alpha gene status in primary breast cancer is a predictive marker of the response to anthracycline-based neoadjuvant chemotherapy

This study aimed at evaluating the usefulness of topoisomerase II alpha (TOP2A) for predicting the effect of anthracycline-based neoadjuvant chemotherapy in breast cancer. The TOP2A status was examined using fluorescent in situ hybridization (FISH) in 14 pre-chemotherapeutic breast cancer tissues, and was also assessed by immunohistochemistry (IHC) in 14 pairs of pre- and post-chemotherapeutic breast cancer specimens. TOP2A gene aberration by IHC tended to show a correlation with pathological responses but this was not statistically significant (p=0.060). On the other hand, the low TOP2A/CEP17 ratio correlated with good pathological responses (p=0.012). TOP2A overexpression was not significantly associated with response (p=0.580). Our results thus suggest that the TOP2A/CEP17 ratio may be a useful predictor of the effects of anthracycline-based neoadjuvant chemotherapy in breast cancer.     

Brain abscess and necrotic brain tumor: discrimination with proton MR spectroscopy and diffusion-weighted imaging

BACKGROUND AND PURPOSE: Discriminating pyogenic brain abscesses from cystic or necrotic tumors is sometimes difficult with CT or MR imaging. We compared findings of proton MR spectroscopy ((1)H-MRS) with those of diffusion-weighted imaging to determine which technique was more effective for this differential diagnosis. METHODS: Fourteen patients (necrotic or cystic tumor [n = 7]; pyogenic abscess [n = 7]) who underwent 1.5-T (1)H-MRS and diffusion-weighted imaging and had findings of ring-shaped enhancement after contrast agent administration were enrolled in this study. Diffusion-weighted imaging was performed with a single-shot spin-echo echo-planar pulse sequence (b = 1000 s/mm(2)). The apparent diffusion coefficient and ratio were also measured. RESULTS: Spectra for two patients were unacceptable because of either poor shimming conditions or contamination from neighboring fat. Spectra in three of five patients with abscess had lactate, amino acids (including valine, alanine, and leucine), and acetate peaks; one of the three spectra had an additional peak of succinate. In one patient with abscess treated by antibiotics, only lactate and lipid peaks were detected. Spectra for four of seven patients with cystic or necrotic tumors showed only lactate peaks. Lactate and lipids were found in three patients with tumors. Hyperintensity was seen in all the pyogenic abscess cavities and hypointensity in all the cystic and necrotic tumors on diffusion-weighted images. CONCLUSION: (1)H-MRS and diffusion-weighted imaging are useful for differentiating brain abscess from brain tumor, but the latter requires less time and is more accurate than is (1)H-MRS. (1)H-MRS is probably more limited in cases of smaller peripheral lesions, skull base lesions, and treated abscesses.     

Early rapid growth,early birth: Accelerated fetal growth and spontaneous late preterm birth

The past two decades in the United States have seen a 24% rise in spontaneous late preterm delivery (34–36 weeks) of unknown etiology. This study tested the hypothesis that fetal growth was identical prior to spontaneous preterm (n = 221, median gestational age at birth 35.6 weeks) and term (n = 3706) birth among pregnancies followed longitudinally in Santiago, Chile. The hypothesis was not supported: Preterm‐delivered fetuses were significantly larger than their term‐delivered peers by mid‐second trimester in estimated fetal weight, head, limb, and abdominal dimensions, and they followed different growth trajectories. Piecewise regression assessed time‐specific differences in growth rates at 4‐week intervals from 16 weeks. Estimated fetal weight and abdominal circumference growth rates slowed at 20 weeks among the preterm‐delivered, only to match and/or exceed their term‐delivered peers at 24–28 weeks. After an abrupt growth rate decline at 28 weeks, fetuses delivered preterm did so at greater population‐specific sex and age‐adjusted birth weight percentiles than their peers from uncomplicated pregnancies (P < 0.01). Growth rates predicted birth timing: one standard score of estimated fetal weight increased the odds ratio for late preterm birth from 2.8 prior to 23 weeks, to 3.6 (95% confidence interval, 1.82–7.11, P < 0.05) between 23 and 27 weeks. After 27 weeks, increasing size was protective (OR: 0.56, 95% confidence interval, 0.38–0.82, P = 0.003). These data document, for the first time, a distinctive fetal growth pattern across gestation preceding spontaneous late preterm birth, identify the importance of mid‐gestation for alterations in fetal growth, and add perspective on human fetal biological variability. Am. J. Hum. Biol., 2009. © 2008 Wiley‐Liss, Inc.     

Targeted and untargeted CD137L fusion proteins for the immunotherapy of experimental solid tumors.

Introduction: CD137L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to T cells. In this study, two novel CD137L fusion proteins were produced and compared with the CD137 agonist antibody 2A. Materials and Methods: Murine CD137L was linked to the COOH terminus of either the Fc fragment of immunoglobulin (untargeted version) or TNT-3 (targeted version), an antibody that binds to necrotic regions of tumors. Groups of mice bearing established Colon 26 tumors were then treated daily x 5 with each fusion protein or 2A to determine their immunotherapeutic potential. RESULTS: Both fusion proteins retained CD137L activity in vitro and TNT-3/CD137L showed tumor-binding activity by biodistribution analysis in tumor-bearing mice. The fusion proteins also produced similar responses in vivo at the 1 nmol per dose range and showed a 60% (TNT-3/CD137L) or 40% (Fc/CD137L) survival of treated mice at 150 days after tumor implantation, similar to the effects of 2A. Morphologic and immunohistochemical analyses showed massive central necrosis and infiltration of granzyme B-positive cells in necrotic areas and viable peripheral regions of treated tumors. Finally, cell depletion studies showed that CD137L-mediated tumor regression was CD8(+) T cell dependent. CONCLUSIONS: From these studies, it was determined that both targeted and untargeted CD137L fusion proteins showed effective antitumor activity, but that the targeted version was more potent. Therefore, the use of the natural CD137 ligand is a promising approach to the treatment of solid tumors by virtue of its ability to produce physiologic costimulation within the tumor, limiting side effects often seen with agonist antibody therapies.