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Endogenous creatinine clearance (Ccr) is widely accepted as an estimate of glomerular filtration rate (GFR), the best overall biomarker of kidney function. However, current common methods of measuring creatinine are not sensitive enough for mouse plasma. Accordingly, we here report a new method of measuring creatinine by liquid chromatography tandem mass spectrometry (LC-MS/MS) using deuterated [2H3]-creatinine as an internal standard. The assay requires 10 microl or less of plasma or urine, and is eight times more sensitive than high-performance liquid chromatography. The reproducibility of the assay of replicates is approximately +/-10%. The plasma creatinine levels of wild type male C57BL/6J mice obtained by LC-MS/MS are 0.076+/-0.002 mg/dl (n=65). To estimate daily urinary creatinine excretion for calculating Ccr, we collected urine from mice housed in metabolic cages, and combined this with washes from the cage internal surfaces. Creatinine in the wash varies from 4 to 67% of the total daily urinary creatinine excretion (typically approximately 400 microg/day). Ccr obtained by LC-MS/MS was 329+/-17 microl/min, which is indistinguishable from GFR measured by using fluorescein isothiocyanate-inulin. The LC-MS/MS method is sensitive, specific, simple, fast, and inexpensive; it is suitable for estimating GFR in conscious mice or other small animals. As it allows repeated measurements in the same animals, it facilitates detection of subtle differences or changes in renal function.

Background:In this study, we evaluated the ability of gene expression profiles to predict chemotherapy response and survival in triple-negative breast cancer (TNBC).Methods:Gene expression and clinical–pathological data were evaluated in five independent cohorts, including three randomised clinical trials for a total of 1055 patients with TNBC, basal-like disease (BLBC) or both. Previously defined intrinsic molecular subtype and a proliferation signature were determined and tested. Each signature was tested using multivariable logistic regression models (for pCR (pathological complete response)) and Cox models (for survival). Within TNBC, interactions between each signature and the basal-like subtype (vs other subtypes) for predicting either pCR or survival were investigated.Results:Within TNBC, all intrinsic subtypes were identified but BLBC predominated (55–81%). Significant associations between genomic signatures and response and survival after chemotherapy were only identified within BLBC and not within TNBC as a whole. In particular, high expression of a previously identified proliferation signature, or low expression of the luminal A signature, was found independently associated with pCR and improved survival following chemotherapy across different cohorts. Significant interaction tests were only obtained between each signature and the BLBC subtype for prediction of chemotherapy response or survival.Conclusions:The proliferation signature predicts response and improved survival after chemotherapy, but only within BLBC. This highlights the clinical implications of TNBC heterogeneity, and suggests that future clinical trials focused on this phenotypic subtype should consider stratifying patients as having BLBC or not.

Two triple immunization vaccine regimens with adenoviral vectors with E1 deleted expressing Gag of human immunodeficiency virus type 1 were tested for induction of T-and B-cell-mediated-immune responses in mice and in nonhuman primates. The vaccine carriers were derived from distinct serotypes of human and simian adenoviruses that fail to elicit cross-neutralizing antibodies expected to dampen the effect of booster immunizations. Both triple immunization regimens induced unprecedented frequencies of gamma interferon-producing CD8؉ T cells to Gag in mice and monkeys that remained remarkably stable over time. In addition, monkeys developed Gag-specific interleukin-2-secreting T cells, presumably belonging to the CD4 ؉ T-cell subset, and antibodies to both Gag and the adenoviral vaccine carriers.More than 40 million humans are presently infected with human immunodeficiency virus type 1 (HIV-1), and most live in developing countries with no access to antiretroviral drug treatment. A vaccine to HIV-1, which would provide the only cost effective and thus viable option to stem the epidemic, remains elusive. One of the most promising vaccine candidates, now in phase 1 clinical trials, is based on an E1 deletion human serotype 5 adenoviral (Ad) recombinant (AdHu5) (5, 15). However, preexposure to this ubiquitous human pathogen resulting in the circulation of neutralizing antibodies (NAs), which are found in many adult humans (6), can strongly limit the efficacy of an AdHu5 vaccine carrier (7).To circumvent preexisting immunity and to broaden the available repertoire of Ad vaccine carriers suitable for booster immunizations, we developed a panel of E1 deletion Ad recombinants based on chimpanzee isolates (3). We previously reported on the AdC68 vector (7) and now extend these studies to two additional chimpanzee isolates termed AdC6 and AdC7. E1 deletion vectors based on molecular clones of AdC6 and AdC7 virus were generated to express a truncated form of Gag of HIV-1. They were tested in combination with an E1 deletion AdHu5 vector encoding the same transgene product for induction of T-cell-mediated immune responses in mice and nonhuman primates (NHPs). A triple immunization protocol with sequential use of heterologous Ad vaccine carriers was shown to induce exceedingly potent CD8 ϩ and CD4 ϩ T-cell responses as well as antibodies to Gag in mice as well as in NHPs. MATERIALS AND METHODSExperimental animals. Two-to three-year-old Chinese rhesus macaques purchased from Covance Research Products (Alice, Texas) were housed in the Nonhuman Primate Facility of the Division of Medical Genetics of the University of Pennsylvania. BALB/c mice were purchased from Charles River (Boston, Mass.) and housed at the Animal Facility of The Wistar Institute.Ad recombinants. Vectors were constructed from molecular clones, propagated on HEK 293 cells, purified, and titrated as described previously (6, 11; J. M. Wilson, submitted for publication). Protein expression was confirmed by Western blot analysis (data not shown). The virus particle (...

Summary Background Chronic antigen exposure and/or ageing increases the frequency of T-box expressed in T cells (T-bet)-expressing B-lymphocytes in mice. The frequency and significance of B-cell T-bet expression during chronic hepatitis C (HCV) infection in human subjects has never been described. Methods Healthy controls, cirrhotic and noncirrhotic HCV-infected patients, and non-HCV patients with cirrhosis were recruited. Peripheral blood mononuclear cells were phenotyped for expression of T-bet and related markers by flow cytometry. In a subset of patients who underwent antiviral therapy and were cured of HCV infection (sustained virological response), the dynamics of T-bet expression in B cells was monitored. After cure, convalescent B cells were tested for T-bet expression after re-exposure to infected plasma or recombinant HCV proteins. Results Forty-nine patients including 11 healthy donors, 30 hepatitis C-infected individuals (nine with liver cancer, 13 with cirrhosis, eight without cirrhosis) and eight patients with cirrhosis due to non-HCV-related cause were recruited. We found that B cells in patients with chronic HCV exhibited increased frequency of T-bet+ B cells relative to noninfected individuals (median 11.5% v. 2.2%, P<.0001) but that there were no significant differences between noncirrhotic, cirrhotic and cancer-bearing infected individuals. T-Bet+ B cells expressed higher levels of CD95, CXCR3, CD11c, CD267 and FcRL5 compared to T-bet− B cells and predominantly exhibit a tissue-like memory CD27− CD21− phenotype independent of HCV infection. T-bet+ B cells in HCV-infected patients were more frequently class-switched IgD−IgG+ (40.4% vs. 26.4%, P=.012). Resolution of HCV infection with direct-acting antiviral (DAA) therapy leads to a marked reduction in the frequency of T-bet+ B cells (median 14.1% pretreatment v. 6.7% end of treatment v. 6.1% SVR12, P≤.01). Re-exposure of convalescent (cured) B cells to viremic plasma and recombinant HCV E2 protein led to re-expression of T-bet. Conclusion Chronic antigenemia in chronic HCV infection induces and maintains an antigen-specific T-bet+ B cell. These B cells share markers with tissue-like memory B cells. Antigen-driven T-bet expression may be a critical suppressor of B-cell activation in chronic HCV infection.