Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Development and characterization of a whole-cell radioligand binding assay for [125I]gp120 of HIV-1.

The binding of HIV-1 envelope glycoprotein, gp120, to the CD4 receptor is an important step in productive infection. The development of agents which interrupt this binding phenomenon should be of therapeutic interest. The present study characterizes a whole cell gp120/CD4 radioligand binding assay (radioligand binding assay) modified for use in a high volume screening format. Modifications include the use of human CD4 receptor stably expressed in a Chinese hamster ovary cell line and the gentle fixation (paraformaldehyde) of the CD4 receptor just prior to assay. Binding of [125I]gp120 to fixed CD4 was of high affinity (KD = 6 nM), saturable, reversible, and specific. The kinetics of binding were identical to those of viable (non-fixed) CD4 receptor. [125I]gp120 binding was inhibited by unlabeled recombinant gp120, soluble CD4, and the anti-CD4 monoclonals OKT4A and LEU3A. A number of compounds reported to inhibit gp120 binding and/or gp120 induced syncytium formation were also active in this assay. This modified radioligand binding assay was developed to initiate a rational and extensive screening program to assist in the identification of potential chemotherapeutic agents based on their ability to inhibit gp120 binding to host cells.     

Alumina-alumina artificial hip joints. Part II: characterisation of the wear debris from in vitro hip joint simulations

Until recently it was not possible to reproduce clinically relevant wear rates and wear patterns in in vitro hip joint simulators for alumina ceramic-on-ceramic hip prostheses. The introduction of microseparation of the prosthesis components into in vitro wear simulations produced clinically relevant wear rates and wear patterns for the first time. The aim of this study was to characterise the wear particles generated from standard simulator testing and microseparation simulator testing of hot isostatically pressed (HIPed) and non-HIPed alumina ceramic-on-ceramic hip prostheses, and compare these particles to those generated in vivo. Standard simulation conditions produced wear rates of approximately 0.1 mm3 per million cycles for both material types. No change in surface roughness was detected and very few wear features were observed. In contrast, when microseparation was introduced into the wear simulation, wear rates of between 1.24 (HIPed) and 1.74 mm3 per million cycles (non-HIPed) were produced. Surface roughness increased and a wear stripe often observed clinically on retrieved femoral heads was also reproduced. Under standard simulation conditions only nanometre-sized wear particles (2-27.5 nm) were observed by TEM, and it was thought likely that these particles resulted from relief polishing of the alumina ceramic. However, when microseparation of the prosthesis components was introduced into the simulation, a bi-modal distribution of particle sizes was observed. The nanometre-sized particles produced by relief polishing were present (1-35nm). however, larger micrometre-sized particles were also observed by both transmission electron microscopy (TEM) (0.021 microm) and scanning electron microscopy (SEM) (0.05-->10 microm). These larger particles were thought to originate from the wear stripe and were produced by trans-granular fracture of the alumina ceramic. In Part I of this study, alumina ceramic wear particles were isolated from the periprosthetic tissues from around Mittelmeier ceramic-on-ceramic hip prostheses. Characterisation of the particles by TEM and SEM revealed a bi-modal size distribution. SEM analysis revealed particles in the 0.05-3.2 microm size range. and TEM revealed particles in the 5-90 nm size range, indicating that microseparation of the prosthesis components may be a common event in vivo. This study (Part II) has revealed that the introduction of microseparation of the prosthesis components during the swing phase of the wear simulation reproduced clinically relevant wear rates, wear patterns and wear particles in in vitro hip joint simulators.     

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.      

High-sensitivity PCR detection of parvovirus B19 in plasma

Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10(4) genome equivalents/ml may not be infectious, while those greater than 10(7)/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10(4) IU/ml (6.5 x 10(3) genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 x 10(3) IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 x 10(3) to 2 x 10(5) IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 +/- 17.5 IU/ml (mean +/- standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.     

Evidence for an Association of the Dopamine D5 Receptor Gene on Age at Onset of Attention Deficit Hyperactivity Disorder

The purpose of this study was to determine whether the single nucleotide polymorphisms (SNPs) within candidate genes for attention deficit hyperactivity disorder (ADHD) are associated with the age at onset for ADHD. One hundred and forty-three SNPs were genotyped across five candidate genes ( DRD5 , SLC6A3 , HTR1B , SNAP25 , DRD4 ) for ADHD in 229 families with at least one affected offspring. SNPs with the highest estimated power to detect an association with age at onset were selected for each candidate gene, using a power-based screening procedure that does not compromise the nominal significance level. A time-to-onset analysis for family-based samples was performed on these SNPs to determine if an association exists with age at onset for ADHD. Seven consecutive SNPs surrounding the D5 dopamine receptor gene ( DRD5 ), were associated with the age at onset for ADHD; FDR adjusted q-values ranged from 0.008 to 0.023. This analysis indicates that individuals with the risk genotype develop ADHD earlier than individuals with any other genotype. A haplotype analysis across the 6 significant SNPs that were in linkage disequilibrium with one another, CTCATA , was also found to be significant (p-value = 0.02). We did not observe significant associations with age at onset for the other candidate loci tested. Although definitive conclusions await independent replication, these results suggest that a variant in DRD5 may affect age at onset for ADHD.     

New complementary perspectives for inpatient physical function assessment: matched clinician-report and patient-report short form measures from the PROMIS adult physical function item bank

Quality of Life Research - To develop two item content-matched, precise, score-level targeted inpatient physical function (PF) short form (SF) measures: one clinician-reported, one...     

Stochastic programming for outpatient scheduling with flexible inpatient exam accommodation

This study is concerned with the determination of an optimal appointment schedule in an outpatient-inpatient hospital system where the inpatient exams can be cancelled based on certain rules while the outpatient exams cannot be cancelled. Stochastic programming models were formulated and solved to tackle the stochasticity in the procedure durations and patient arrival patterns. The first model, a two-stage stochastic programming model, is formulated to optimize the slot size. The second model further optimizes the inpatient block (IPB) placement and slot size simultaneously. A computational method is developed to solve the second optimization problem. A case study is conducted using the data from Magnetic Resonance Imaging (MRI) centers of Lahey Hospital and Medical Center (LHMC). The current schedule and the schedules obtained from the optimization models are evaluated and compared using simulation based on FlexSim Healthcare. Results indicate that the overall weighted cost can be reduced by 11.6% by optimizing the slot size and can be further reduced by an additional 12.6% by optimizing slot size and IPB placement simultaneously. Three commonly used sequencing rules (IPBEG, OPBEG, and a variant of ALTER rule) were also evaluated. The results showed that when optimization tools are not available, ALTER variant which evenly distributes the IPBs across the day has the best performance. Sensitivity analysis of weights for patient waiting time, machine idle time and exam cancellations further supports the superiority of ALTER variant sequencing rules compared to the other sequencing methods. A Pareto frontier was also developed and presented between patient waiting time and machine idle time to enable medical centers with different priorities to obtain solutions that accurately reflect their respective optimal tradeoffs. An extended optimization model was also developed to incorporate the emergency patient arrivals. The optimal schedules from the extended model show only minor differences compared to those from the original model, thus proving the robustness of the scheduling solutions obtained from our optimal models against the impacts of emergency patient arrivals.

     

The Sydney AFF Score: A Simple Tool to Distinguish Females Presenting With Atypical Femur Fractures Versus Typical Femur Fractures

Atypical femur fractures (AFF) are a rare but serious complication of long-term bisphosphonate use. Although clearly defined by ASBMR criteria, a proportion of patients with AFFs may go unrecognized and the use of qualitative fracture criteria may lead to uncertainty in AFF diagnosis, with significant therapeutic implications. A score that rapidly and accurately identifies AFFs among subtrochanteric femur fractures using quantitative, measurable parameters is needed. In a retrospective cohort of 110 female patients presenting with AFFs or typical femur fractures (TFFs), multiple logistic regression and decision tree analysis were used to develop the Sydney AFF score. This score, based on demographic and femoral geometry variables, uses three dichotomized independent predictors and adds one point for each: (age ≤80 years) + (femoral neck width <37 mm) + (lateral cortical width at lesser trochanter ≥5 mm), (score, 0 to 3). In an independent validation set of 53 female patients at a different centre in Sydney, a score ≥2 demonstrated 73.3% sensitivity and 69.6% specificity for AFF (area under the receiver-operating characteristic curve [AUC] 0.775, SE 0.063) and remained independently associated with AFF after adjustment for bisphosphonate use. The Sydney AFF score provides a quantitative means of flagging female patients with atraumatic femur fractures who have sustained an AFF as opposed to a TFF. This distinction has clear management implications and may augment current ASBMR diagnostic criteria. © 2021 American Society for Bone and Mineral Research (ASBMR).