CD4 coating, but not CD4 depletion, is a predictor of efficacy with primatized monoclonal anti-CD4 treatment of active rheumatoid arthritis

OBJECTIVE: Double blind studies were conducted with the anti-CD4 monoclonal antibody (Mab) keliximab in patients with active, stable rheumatoid arthritis (RA), to confirm preliminary evidence of efficacy and safety from open. uncontrolled studies. METHODS: We enrolled 136 and 186 patients into 2 consecutive, randomized, double blind trials, with similar populations [apart from inclusion of disease modifying antirheumatic drug (DMARD)-na?ve patients in Study 2]. Patients received 4 weeks intravenous placebo or keliximab [40, 80, 120, or 140 mg twice weekly (bw), or 240 mg once weekly (ow)].The primary endpoint was the American College of Rheumatology (ACR) 20 response criteria, one week after the end of treatment. RESULTS: ACR 20 response rates in Study I were 19%, 42%, 51%*, and 69%* (*p < 0.05 compared to placebo), with placebo, 40, 80, or 140 mg keliximab bw, respectively. The response rates in Study 2 were 30%, 39%, 46% and 47% with placebo, 80 or 120 mg bw, or 240 mg keliximab ow, respectively. In the 2 studies, there was a dose dependent increase in peripheral blood CD4+ T cell coating with keliximab, but a different pattern of CD4 depletion was seen. While only 12% of keliximab treated patients in Study I had CD4 counts below 250 cells/mm3 at the end of the treatment period, 47% fell below this level in Study 2. Clinical response was not correlated with CD4 depletion, but was correlated with CD4+ T cell coating with keliximab. CONCLUSION: Coating of peripheral blood CD4+ T cells with keliximab, but not CD4 depletion, is a determinant of clinical response.     

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.     

Sleep-disordered breathing in alcoholics

Sleep apnea and related disorders contribute to disturbed sleep in abstinent alcoholics. In an earlier report from our group, sleep-disordered breathing was common and increased with age in a cohort of 75 abstinent alcoholics. We now report an extension of the previous work that includes studies of an additional 103 abstinent alcoholics undergoing treatment for alcoholism (total sample = 188) and a comparison group of 87 normal subjects. The presence and severity of sleep-disordered breathing was assessed with polysomnography. Among the alcoholics, sleep-disordered breathing (defined as 10 or more apneas plus hypopneas per hour of sleep) was present in 3% of 91 subjects under age 40, 17% of 83 subjects age 40 to 59, and 50% of 14 subjects age 60 or over. Subjects with sleep-disordered breathing were more likely to be male and had more severe sleep disruption and nocturnal hypoxemia and more complaints related to daytime sleepiness than subjects without sleep-disordered breathing. In a multiple linear regression analysis, age and body mass index were significant predictors of the presence of sleep-disordered breathing, whereas smoking history and duration of heavy drinking were not predictors after controlling for the effects of age and body mass index. Our findings suggest that sleep-disordered breathing contributes significantly to sleep disturbance in a substantial proportion of older alcoholics and that symptomatic sleep-disordered breathing increases with age in alcoholics. Sleep-disordered breathing, when combined with existing cardiovascular risk factors, may contribute to adverse health consequences in alcoholics.     

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.     

Validating drug repurposing signals using electronic health records: a case study of metformin associated with reduced cancer mortality

Objectives Drug repurposing, which finds new indications for existing drugs, has received great attention recently. The goal of our work is to assess the feasibility of using electronic health records (EHRs) and automated informatics methods to efficiently validate a recent drug repurposing association of metformin with reduced cancer mortality.Methods By linking two large EHRs from Vanderbilt University Medical Center and Mayo Clinic to their tumor registries, we constructed a cohort including 32 415 adults with a cancer diagnosis at Vanderbilt and 79 258 cancer patients at Mayo from 1995 to 2010. Using automated informatics methods, we further identified type 2 diabetes patients within the cancer cohort and determined their drug exposure information, as well as other covariates such as smoking status. We then estimated HRs for all-cause mortality and their associated 95% CIs using stratified Cox proportional hazard models. HRs were estimated according to metformin exposure, adjusted for age at diagnosis, sex, race, body mass index, tobacco use, insulin use, cancer type, and non-cancer Charlson comorbidity index.Results Among all Vanderbilt cancer patients, metformin was associated with a 22% decrease in overall mortality compared to other oral hypoglycemic medications (HR 0.78; 95% CI 0.69 to 0.88) and with a 39% decrease compared to type 2 diabetes patients on insulin only (HR 0.61; 95% CI 0.50 to 0.73). Diabetic patients on metformin also had a 23% improved survival compared with non-diabetic patients (HR 0.77; 95% CI 0.71 to 0.85). These associations were replicated using the Mayo Clinic EHR data. Many site-specific cancers including breast, colorectal, lung, and prostate demonstrated reduced mortality with metformin use in at least one EHR.Conclusions EHR data suggested that the use of metformin was associated with decreased mortality after a cancer diagnosis compared with diabetic and non-diabetic cancer patients not on metformin, indicating its potential as a chemotherapeutic regimen. This study serves as a model for robust and inexpensive validation studies for drug repurposing signals using EHR data.     

In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C-2 symmetrical inhibitors of HIV type 1 protease.

Protease inhibitors are another class of compounds for treatment of human immunodeficiency virus (HIV)-caused disease. The emergence of resistance to the current anti-HIV drugs makes the determination of potential resistance to protease inhibitors imperative. Here we describe the isolation of an HIV type 1 (HIV-1) resistant to an HIV-protease inhibitor. Serial passage of HIV-1 (strain RF) in the presence of the inhibitor, [2-pyridylacetylisoleucylphenylalanyl-psi (CHOH)]2 (P9941), failed to yield a stock of virus with a resistance phenotype. However, variants of the virus with 6- to 8-fold reduced sensitivity to P9941 were selected by using a combination of plaque assay and endpoint titration. Genetic analysis and computer modeling of the variant proteases revealed a single change in the codon for amino acid 82 (Val-->Ala), which resulted in a protease with lower affinity and reduced sensitivity to this inhibitor and certain, but not all, related inhibitors.     

Electrocardiographic manifestations and clinical significance of atrioventricular nodal alternating Wenckbach periods

Atrioventricular nodal alternating Wenckebach periods were defined as episodes of 2:1 atrioventricular block in which there was a gradual increase in transmission intervals of conducted beats ending in two or three consecutively blocked atrial impulses. This is one of the mechanisms whereby 2:1 atrioventricular block progresses into 3:1 or 4:1 atrioventricular block. Alternating Wenckebach periods appear during rapid atrial pac,ng (even in the absence of depressed atrioventricular nodal function), provided that the atria can be captured at a rate fast enough to allow for the occurrence of this phenomenon. Treatment of atrial flutter with digoxin and quinidine produces alternating Wenckebach's periods, with associated electrocardiographic changes specific for the type of drug given. In patients with "atrial tachycardia with atrioventricular block" due to digitalis intoxication or with primary disease of the conducting system or with acute myocardial infarction, there are coexisting severe arrhythmias and clinical symptoms requiring almost immediate pharmacologic or electrical therapy. We conclude that atrioventricular nodal alternating Wenckebach's periods are common and frequentyly transient and that they occur in a variety of clinical conditions, most of which are benign; however, contrary to what is commonly accepted, some episodes appear in clinical settings requiring prompt pharmacologic or electrical treatment.     

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.