The relationship of human platelet density to platelet age: platelet population labeling by monoamine oxidase inhibition

The relationship between platelet density and platelet age appears to vary between species with relatively few labeling studies in humans reported. In this study, irreversible monoamine oxidase (MAO) inhibitors were used to biochemically label the circulating platelet population in 15 humans. Platelet samples were then isolated during the 15 days after drug ingestion. The platelets were separated by density on continuous linear Percoll gradients and the density distributions were divided into five fractions containing approximately equal numbers of platelets. Baseline MAO activity was strongly correlated with platelet density. Twenty-four hours after a single dose of tranylcypromine, platelet MAO activities in the density subpopulations were reduced to 14% to 17% of the baseline values. During the first five days after inhibition, the rates of recovery of MAO activity (percentage per day) were inversely proportional to platelet density. The recovery rates in the two most dense fractions were initially slow but increased after five days. Percentage recovery of MAO activity in the least dense fraction was significantly greater than the percentage recovery in the most dense fraction on days 2, 3, 5, and 8 (P less than .01, sign test). These results support the hypothesis that normal human platelets show a small increase in density with age, but they do not exclude the additional possibility that human platelet lifespan is positively correlated with platelet density.     

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Infection and Malignancy Outweigh Cardiovascular Mortality in Kidney Transplant Recipients: Post Hoc Analysis of the FAVORIT Trial

Objective

Now that long-term survival after successful renal transplantation is no longer limited by excessive cardiovascular risk, the primary care physician should consider that infection and malignancy are leading noncardiovascular causes of death even in the recipient with diabetes.

Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.     

Assessing the delivery of neutrophils to tissues in neutropenia

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.     

Influence of Renal Function on the Pharmacokinetics,Pharmacodynamics, Efficacy,and Safety of Non–Vitamin K Antagonist Oral Anticoagulants

With the growing integration of non–vitamin K antagonist oral anticoagulants (NOACs) into clinical practice, questions have arisen regarding their use in special populations, including groups that may have been underrepresented in clinical trials. Patients with renal impairment, particularly in the lower echelons of renal function, are one such group. In an effort to elucidate the current evidence regarding the use of NOACs in patients with renal impairment, a systematic assessment of the literature was performed. The MEDLINE database was interrogated for studies and analyses evaluating the influence of renal function on the pharmacokinetics, pharmacodynamics, efficacy, and safety of NOACs published from January 1, 2000, through August 2, 2017. The 82 relevant publications retrieved highlight the diversity in the NOAC class regarding the impact of renal function on drug clearance, drug exposures, and clinical trial outcomes. In several large clinical trials, subgroup analyses revealed no significant differences when patients were stratified by creatinine clearance as a measure of renal function. Efficacy findings, in particular, were largely aligned with the overall population in the included studies. However, relative risks of bleeding were shown to vary, sometimes driven by changes in bleeding event rates in the comparator arm (eg, warfarin, enoxaparin). With few exceptions, minimal influence of mild renal impairment was observed on the relative efficacy and safety of NOACs. Taken together, the evidence suggests that the presence of renal impairment merits careful consideration of anticoagulant choice but should not deter physicians from appropriate use of NOACs.     

Early PREdiction of sepsis using leukocyte surface biomarkers: the ExPRES-sepsis cohort study

Purpose

Reliable biomarkers for predicting subsequent sepsis among patients with suspected acute infection are lacking. In patients presenting to emergency departments (EDs) with suspected acute infection, we aimed to evaluate the reliability and discriminant ability of 47 leukocyte biomarkers as predictors of sepsis (Sequential Organ Failure Assessment score?≥?2 at 24 h and/or 72 h following ED presentation).

Methods

In a multi-centre cohort study in four EDs and intensive care units (ICUs), we standardised flow-cytometric leukocyte biomarker measurement and compared patients with suspected acute infection (cohort-1) with two comparator cohorts: ICU patients with established sepsis (cohort-2), and ED patients without infection or systemic inflammation but requiring hospitalization (cohort-3).

Results

Between January 2014 and February 2016, we recruited 272, 59 and 75 patients to cohorts 1, 2, and 3, respectively. Of 47 leukocyte biomarkers, 14 were non-reliable, and 17 did not discriminate between the three cohorts. Discriminant analyses for predicting sepsis within cohort-1 were undertaken for eight neutrophil (cluster of differentiation antigens (CD) CD15; CD24; CD35; CD64; CD312; CD11b; CD274; CD279), seven monocyte (CD35; CD64; CD312; CD11b; HLA-DR; CD274; CD279) and a CD8 T-lymphocyte biomarker (CD279). Individually, only higher neutrophil CD279 [OR 1.78 (95% CI 1.23–2.57); P?=?0.002], higher monocyte CD279 [1.32 (1.03–1.70); P?=?0.03], and lower monocyte HLA-DR [0.73 (0.55–0.97); P?=?0.03] expression were associated with subsequent sepsis. With logistic regression the optimum biomarker combination was increased neutrophil CD24 and neutrophil CD279, and reduced monocyte HLA-DR expression, but no combination had clinically relevant predictive validity.

Conclusions

From a large panel of leukocyte biomarkers, immunosuppression biomarkers were associated with subsequent sepsis in ED patients with suspected acute infection.

Clinical trial registration

NCT02188992.