Anti-HBs Cellular Immune Response in Kidney Recipients before and 4 Months after Transplantation

Patients with renal failure represent a population at risk for hepatitis B, since only 50 to 60% of them develop protective humoral responses after vaccination. As this could be due to an altered regulation of cellular immune responses, the objectives of the present study were to evaluate the proliferative abilities of lymphocytes from patients with chronic renal failure after stimulation in vitro with a mitogen (pokeweed mitogen [PWM]) or HBsAg. In order to differentiate between the immunodeficiency associated with renal failure and that due to immunosuppression posttransplantation, the same subjects were tested before and 4 months after kidney transplantation. The lymphoproliferation assay used was performed by flow cytometry, which is based on sequential analysis of the cell cycle and which allows analysis of cytokine production. Serologically, the group of 36 patients tested comprised 22% nonresponders, 30% poor responders, and 48% responders. Lymphocyte growth was observed for all patients after stimulation with PWM, indicating that these cells had the capacity to proliferate in vitro. The level of lymphoproliferation in response to PWM was significantly reduced after transplantation, yet both before and after transplantation, all serologic nonresponders developed cellular responses to at least two vaccines. No correlation between humoral and cellular responses was shown. Proliferating cells were lymphocytes, which mostly secreted interleukin 4 (IL-4) and IL-10 for the three serologic groups. This study suggests that even when repeated vaccination fails to induce significant antibody levels in patients with renal failure, specific HBs cellular responses develop, and these may prove to be efficient in protecting these patients against hepatitis B.     

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium.

Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.     

Inclusion of Performance Parameters and Patient Context in the Clinical Practice Guidelines for Heart Failure

BackgroundTo facilitate evidence-based medicine (EBM) on an individual level, it may be important for clinical practice guidelines (CPGs) to incorporate the performance parameters of diagnostic studies and therapeutic interventions (such as likelihood ratio and absolute benefit or harm), and to incorporate relevant patient contexts that may influence decision-making. We sought to determine the extent to which heart failure CPGs currently incorporate this information.MethodsWe reviewed the American College of Cardiology Foundation/American Heart Association (ACCF/AHA) 2013 Heart Failure CPG, the 2017 ACCF/AHA/HFSA update, and European Society of Cardiology (ESC) 2016 Heart Failure CPG. We abstracted variables for each CPG recommendation from the following domains: quality of evidence, strength of recommendation, diagnostic and therapeutic performance parameters, and patient context.ResultsWe examined 169 recommendations from the ACCF/AHA 2013 CPGs and 2017 update and 187 recommendations from the 2016 ESC CPGs. Performance parameters for diagnostic studies (2013 ACCF/AHA: 13%; 2017 ACCF/AHA/HFSA update: 0%; 2016 ESC: 0%) and therapeutic interventions (2013 ACCF/AHA: 65%; 2017 ACCF/AHA/HFSA update: 64%; 2016 ESC: 16%) were not commonly included in CPGs. Patient context was included in about half of ACCF/AHA recommendations and a quarter of ESC recommendations.ConclusionsThe majority of recommendations from heart failure CPGs lack information on diagnostic and therapeutic performance parameters and patient context. Given the importance of these components to effectively implement EBM, particularly for a heterogeneous heart failure population, innovative strategies are needed to optimize CPGs so they provide comprehensive yet succinct recommendations that can improve population-level outcomes and ensure optimal patient-centered care.     

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.     

Caregiver Nutritional Health Outcomes of the Simple Suppers Study: Results from a 10 Week,Two-Group Quasi-Experimental Family Meals Intervention

Individuals from racial minority backgrounds, especially those in low income situations, are at increased risk for obesity. Family meals positively impact child nutritional health; however, there is limited evidence examining the impact on caregivers, particularly racial minority and income-restricted individuals. The objective of this intervention study was to determine the effect of Simple Suppers, a 10 week family meals program, on caregiver diet and nutrition outcomes. Intervention versus waitlist control participants were compared from baseline (T0) to post-intervention (T1). In addition, intervention participants were assessed at a 10 week follow-up time point (T2). This study was a two-group quasi-experimental intervention trial. Lessons (10 total) were delivered on a weekly basis for 90 min. Data were collected from intervention and waitlist control participants at T0 and T1, and intervention participants at T2. After baseline (T0) data collection, families enrolled in the immediate upcoming session of Simple Suppers (intervention group) or waited for 10 weeks (waitlist control group) to begin the program. Participants were caregivers of children ages 4–10 years. This study was conducted in a faith-based community center for underserved families in Columbus, Ohio. Primary outcomes were: diet quality assessed by Healthy Eating Index (HEI) total and component scores, and total energy intake (kcal/day); body mass index (BMI) (kg/m²), waist circumference (cm), systolic and diastolic blood pressure (BP) (mmHG); and self-efficacy for having healthy meals and menu planning (both scalar). The impact of the intervention (T0:T1) was assessed using generalized mixed-effects linear regression models. Maintenance of change in study outcomes among intervention participants (T1:T2) was examined with paired t-tests. 109 caregivers enrolled in this study. The retention rate at T1 was 90% (i.e., 98 participants). 56 of 68 intervention participants completed T2, resulting in a retention rate of 82%. Almost all (99%) were female, 61% were Black, and 50% were between 31 and 40 years old. In total, 40% had low income and 37% had low or very low food security. At T1, intervention vs. waitlist controls had a lower daily energy intake (p = 0.04), but an HEI-2010 component score for fatty acids (adequacy) that was lower indicating a lower dietary intake of fatty acids (p = 0.02), and a component score for empty calories (moderation) that was significantly lower indicating a higher intake of empty calorie foods (p = 0.03). At T1, intervention vs. waitlist controls also had a lower BMI (p < 0.001) and systolic BP (p = 0.04), and higher self-efficacy (p = 0.03). There were no group differences in other outcomes. At T2, intervention participants maintained the changes in daily energy intake, BMI, systolic BP, and self-efficacy that improved during the intervention period. There was no change (improvement) in the component score for fatty acids; however, the component score for empty calories significantly improved (p = 0.02). Engagement in the Simple Suppers program led to improvements in caregivers’ daily caloric intake, weight status, systolic blood pressure, and self-efficacy for family meals. Future research should further explore the dietary and nutritional health benefits of family meals among caregivers at the highest risk for obesity.     

Using bar-code technology and medication observation methodology for safer medication administration.

PURPOSE: The implementation of a multidisciplinary approach to systematically decrease medication errors through the use of observation methodology and the deployment of electronic medication administration records (EMARs) and bar-coded-medication administration (BCMA) is described. SUMMARY: For a consistent and reliable approach to data collection, a direct-observation technique was used. The measurement of medication errors using the observation process occurred in two phases-preimplementation and postimplementation. Three inpatient nursing units participated. The control group was a 20-bed cardiac telemetry unit. Intervention group 1 was also a 20-bed cardiac telemetry unit. Intervention group 2 was a 36-bed medical-surgical unit. During the first phase of the study, all three study groups participated in evaluating the medication administration process associated with a manual five-day medication administration record (MAR). A total of 188 errors were reported. The pharmacy, nursing, and information services departments collaborated on the design and deployment of the EMAR and BCMA systems. The systems were implemented in one nursing unit in August 2003, with full implementation on all inpatient units by July 2004. During the second phase of the study, the control group continued to use the manual five-day MAR without a change in the process. Intervention groups 1 and 2 were measured to evaluate the medication administration process using EMAR and BCMA technology. The direct-observation accuracy rate before BCMA was 86.5%; after BCMA, the rate rose to 97%. CONCLUSION: The direct-observation methodology was used to monitor medication administration before and after the deployment of the EMAR and BCMA systems. A 54% reduction of medication administration errors was observed following implementation of a multidisciplinary, collaborative approach to medication safety.     

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

The O⁶-methylguanine-DNA methyltransferase (MGMT) repairs mutagenicand carcinogenic O⁶-alkylguanine in DNA by accepting stoichiometricallythe alkyl group from the base. Although the mouse MGMT is largerthan the human protein because of an additional tetrapeptidesequence, these proteins are 70% homologous. Recombinant MGMTsof the human, the mouse and a mouse mutant with the tetrapeptidedeleted were purified to homogeneity from Escherichia coli.The N-terminal amino acid sequences of these proteins are identicalto those predicted from the nucleotide sequences, and theirmolecular masses deter mined by SDS-PAGE agreed with the predictedvalues. However, the observed isoelectric points of 9.3, 9.2and 9.3, for the human, mouse and mutant mouse proteins respectivelywere significantly different from the values, 8.09, 7.47 and7.49 calculated from the amino acid composition. The extinctioncoefficients E_1%^{280 nm} of human, mouse and mutant mouse proteinwere calculated from amino acid composition to be 18.2, 11.1and 11.3 respectively. These values agree fairly well with calculatedvalues. Human and wild-type mouse MGMTs react with the alkylatedbase in a synthetic DNA substrate poly(dC, dG, m⁶dG) with comparablesecond-order rate constants of 2.2x10⁸ and 3.7x10⁸ 1/M/min at37°C respectively and were inactivated by O⁶-benzylguanineat similar rates. The initial reaction rate (K_in) and rate ofinactivation (k_inact) constants for reaction with the base werecalculated to be 1.8x10^–4 M and 1.4x10^–3/s for thehuman protein, 2.3x10^–4 M and 1.1x10^–3/s for thewild-type mouse protein, and 2.1x10^–4 and 1.4x10^–3/sfor the mutant mouse protein respectively. The MGMTs were inactivatedto the extent of 55—65% after heating at 50°C in 20mMTris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However,in the presence of DNA (200 µg/ml), only 25—35%of the protein was inactivated. Both DNA and RNA inhibited allthree enzymes in a concentration-dependent fashion, althoughDNA was a better inhibitor than RNA. High salt (0.2 M NaCl)inhibited human MGMT by 80%, while the wild-type and the mutantmouse MGMTs were inhibited by 55%. The human protein had higheraffinity for binding to duplex DNAs than the mouse proteins.     

Evaluation of 225Ac for vascular targeted radioimmunotherapy of lung tumors

Several alpha particle emitting radioisotopes have been studied for use in radioimmunotherapy. Ac-225 has the potential advantages of a relatively long half life of 10 days, and a yield of 4 alpha emissions in its decay chain with a total energy release of approximately 28 MeV. A new, 12 coordination site chelating ligand, HEHA, has been chemically modified for coupling to targeting proteins without loss of chelating ability. HEHA was coupled with MAb 201B which binds to thrombomodulin and accumulates efficiently in murine lung. Ac-225 was bound to the HEHA-MAb 201B conjugate and injected into BALB/c mice bearing lung tumor colonies of EMT-6 mammary carcinoma. Biodistribution data at 1 and 4 h postinjection indicated that, as expected, 225Ac was delivered to lung efficiently (> 300% ID/g). The 225Ac was slowly released from the lung with an initial t1/2 = 49 h, and the released 225Ac accumulated in the liver. Injection of free HEHA was only partially successful in scavenging free 225Ac. In addition to the slow release of 225Ac from the chelate, data indicated that decay daughters of 225Ac were also released from the lung. Immediately after organ harvest, the level of 213Bi, the third alpha-decay daughter, was found to be deficient in the lungs and to be in excess in the kidney, relative to equilibrium values. Injected doses of 225Ac MAb 201B of 1.0 microCi, delivering a minimum calculated absorbed dose of about 6 Gy to the lungs, was effective in killing lung tumors, but also proved acutely radiotoxic. Animals treated with 1.0 microCi or more of the 225Ac radioconjugate died of a wasting syndrome within days with a dose dependent relationship. We conclude that the potential for 225Ac as a radioimmunotherapeutic agent is compromised not only by the slow release of 225Ac from the HEHA chelator, but most importantly by the radiotoxicity associated with decay daughter radioisotopes released from the target organ.