Clinical and imaging features in adult patients with bone marrow haemophagocytosis with and without haemophagocytic lymphohistiocytosis: a single-institution experience.

AIM: To evaluate clinical, laboratory, imaging findings, and outcomes of adult patients with bone marrow haemophagocytosis (BMH) who meet the diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH) with those who do not meet the criteria. MATERIALS AND METHODS: A pathology database search was performed from 2009 to 2019 to identify adult patients with BMH. Electronic medical records of 41 patients were reviewed to distinguish those who fulfil the HLH-2004 diagnostic guidelines, which identified 22 patients (11 men; mean age, 53.5 years) who met the criteria (HLH+) and 19 patients (13 men; mean age, 54.7 years) who did not meet the criteria (HLH-). Multi-modality imaging was reviewed to record imaging features. Clinical, laboratory, imaging findings, and outcomes were compared between the two groups using Fisher's exact test and Wilcoxon test. RESULTS: Malignancy (non-Hodgkin's lymphoma) was the major trigger for both groups. 86% of HLH+ and 31% of HLH- patients presented with fever. Compared to the HLH- group, the HLH+ group exhibited higher serum ferritin, triglycerides, and lower fibrinogen levels (p<0.05). Alveolar opacities and hepatosplenomegaly were the most common imaging findings identified in both groups. Median overall survival of HLH+ and HLH- were 123.5 (interquartile range [IQR]: 40.7-681.7 days) and 189 days (IQR: 52-1680 days), respectively. Distribution of imaging features and overall survival did not differ between the groups. CONCLUSION: Malignancy is the major trigger for BMH in both HLH+ and HLH- groups. HLH+ and HLH- groups have similar imaging manifestations or clinical outcomes. Therefore, presence of BMH alone is correlated with high morbidity and mortality.

Tyrosine Kinase Inhibitor Therapy for Brain Metastases in Non-Small-Cell Lung Cancer: A Primer for Radiologists.

Treatment options for patients who develop brain metastases secondary to non-small-cell lung cancer have rapidly expanded in recent years. As a key adjunct to surgical and radiation therapy options, systemic therapies are now a critical component of the oncologic management of metastatic CNS disease in many patients with non-small-cell lung cancer. The aim of this review article was to provide a guide for radiologists, outlining the role of systemic therapies in metastatic non-small-cell lung cancer, with a focus on tyrosine kinase inhibitors. The critical role of the blood-brain barrier in the development of systemic therapies will be described. The final sections of this review will provide an overview of current imaging-based guidelines for therapy response. The utility of the Response Assessment in Neuro-Oncology criteria will be discussed, with a focus on how to use the response criteria in the assessment of patients treated with systemic and traditional therapies.

Phase I and pharmacological study of pazopanib in combination with oral topotecan in patients with advanced solid tumours.

BACKGROUND: This phase I study evaluated the safety, tolerability, maximum tolerated dose (MTD) and pharmacokinetics of two dosing schedules of oral topotecan in combination with pazopanib in patients with advanced solid tumours. METHODS: Stage I of this study was to determine whether there was an impact of pazopanib on topotecan exposure. In stage II, the MTD and safety profile of oral topotecan given weekly on days 1, 8 and 15 in a 28-day cycle; or daily-times-five on days 1-5 in a 21-day cycle, both in combination with daily pazopanib, were explored. RESULTS: In total, 67 patients were enroled. Pazopanib co-administration caused a substantial increase in exposure to total topotecan (1.7-fold) compared with topotecan alone, which is considered clinically relevant. Topotecan had no effect on pazopanib concentrations. Safety findings were consistent with the known profile of both agents. There were three drug-related deaths, liver failure, tumour haemorrhage and myelosuppression. Two patients experienced dose-limiting toxicities (DLTs; hand-foot syndrome, myelosuppression and diarrhoea) on the weekly topotecan schedule and four patients experienced DLTs (myelosuppression) on the daily-times-five topotecan schedule. When combined with pazopanib, 800 mg daily, the recommended doses for oral topotecan are: 8 mg weekly and 2.5 mg daily-times-five. Seven of eight patients with partial response had platinum-resistant ovarian cancer. In addition, 54% of patients had stable disease with 22% stable for 6 months. CONCLUSIONS: Total topotecan exposure is 1.7-fold higher when co-administered with pazopanib. Both schedules of administration were tolerated and would permit further evaluation, especially the weekly schedule.

An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle.

The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl(-) binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small.

Abdominal diameter index and 12-year cardiovascular disease incidence in male bridge and tunnel workers.

BACKGROUND: A recent study has suggested that abdominal diameter index (ADI), that is, the supine sagittal abdominal diameter divided by thigh circumference, may be a better measure of the increased risk of abdominal adiposity for prevalent ischemic cardiovascular disease (CVD) than body mass index or waist circumference. The risk associated with all of these measures is believed to arise from the link between visceral obesity and insulin resistance. METHODS: Male bridge and tunnel workers in New York City without ischemic CVD in the highest and lowest quartiles of ADI (n=218) in a 1993-1994 cross-sectional study of risk factors and prevalent coronary heart disease were administered telephone follow-up questionnaires after 12 years (2005-2006) to assess incident ischemic CVD (new-onset angina, coronary revascularization, myocardial infarction, stroke, peripheral vascular disease and cardiovascular death). RESULTS: In the univariate analysis of 111 participants able to be contacted, study members in the highest quartile vs the lowest quartile of ADI had a significantly increased cumulative incidence of ischemic CVD (Relative risk (RR)=7.9, P=0.002). In a logistic regression analysis controlling for other cardiovascular risk factors including age, smoking, total cholesterol, high-density lipoprotein cholesterol, triglycerides, blood pressure and glucose, ADI lost statistical significance (RR=4.37, P=0.063), suggesting that ADI may be an anthropometric surrogate for these cardiovascular risk factors. CONCLUSIONS: ADI is a powerful anthropometric index for 12-year cumulative incidence of ischemic CVD in working men in New York City.

High resolution imaging of actin filaments in living cells under physilogically relevant conditions using apertureless near-field microscopy.

Living cells were used to demonstrate the potential biological applications of the apertureless SNOM when operating under fluid. An oral epithelial squamous cell carcinoma cell line (H357) was imaged under physiological-like conditions using apertureless SNOM following staining with FITC-Phalloidin which preferentially stains intracellular actin filaments of the cytoskeleton. Compared with simultaneously obtained AFM topographic image, the apertureless SNOM data provides greater detail on these cellular structures and the spatial resolution of the apertureless SNOM fluorescence image appears to be about 100 nm.

Three-dimensional structures reveal multiple ADP/ATP binding modes for a synthetic class of artificial proteins.

The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

Anti-interleukin-5 antibody treatment (mepolizumab) in active eosinophilic oesophagitis: a randomised, placebo-controlled, double-blind trial.

OBJECTIVE: Eosinophilic oesophagitis (EoO) is a clinicopathological condition defined by proton pump inhibitor-refractory oesophageal symptoms combined with oesophageal eosinophilia. The pharmacodynamic effect of mepolizumab (a humanised anti-interleukin-5 monoclonal antibody) in EoO was evaluated. METHODS: Eleven adults with active EoO (>20 peak eosinophil number/high power field (hpf) and dysphagia) were randomised to 750 mg of mepolizumab (n = 5) or placebo (n = 6) and received two intravenous infusions, 1 week apart. Those not in complete remission (<5 peak eosinophil number/hpf) after 8 weeks received two further doses 4 weeks apart, 1500 mg of mepolizumab or placebo. The effect of mepolizumab was assessed clinically, endoscopically, histologically, and via blood and tissue biomarkers. RESULTS: As assessed by immunofluorescence, a marked reduction of mean oesophageal eosinophilia (p = 0.03) was seen in the mepolizumab group (-54%) compared with the placebo group (-5%) 4 weeks after initiation of treatment. No further reduction of eosinophil numbers was observed in response to the two additional infusions in either group. Mepolizumab reduced tenascin C (p = 0.033) and transforming growth factor beta1 (p = 0.05) expression in the oesophageal epithelial layer 13 weeks after initiation of treatment. Clinically, limited improvement of symptoms was seen, although a trend was seen between 4 and 13 weeks after initiation of mepolizumab treatment. Mepolizumab was well tolerated. CONCLUSIONS: Mepolizumab significantly reduced eosinophil numbers in oesophageal tissues in adult patients with active EoO, and changes in the expression of molecules associated with oesophageal remodelling were reversed. Minimal clinical improvement was achieved in a subgroup of patients with EoO. Mepolizumab had an acceptable safety profile, even at the high 1500 mg dose level. TRIAL REGISTRATION NUMBER: NCT00274703.

Role of reactive metabolites in drug-induced hepatotoxicity.

Drugs are generally converted to biologically inactive forms and eliminated from the body, principally by hepatic metabolism. However, certain drugs undergo biotransformation to metabolites that can interfere with cellular functions through their intrinsic chemical reactivity towards glutathione, leading to thiol depletion, and functionally critical macromolecules, resulting in reversible modification, irreversible adduct formation, and irreversible loss of activity. There is now a great deal of evidence which shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, such as acetaminophen, tamoxifen, isoniazid, and amodiaquine. The main theme of this article is to review the evidence for chemically reactive metabolites being initiating factors for the multiple downstream biological events culminating in toxicity. The major objectives are to understand those idiosyncratic hepatotoxicities thought to be caused by chemically reactive metabolites and to define the role of toxic metabolites.

The mechanism of spontaneous oscillatory contractions in skeletal muscle.

Most striated muscles generate steady contractile tension when activated, but some preparations, notably cardiac myocytes and slow-twitch fibers, may show spontaneous oscillatory contractions (SPOC) at low levels of activation. We have provided what we believe is new evidence that SPOC is a property of the contractile system at low actin-myosin affinity, whether caused by a thin-filament regulatory system or by other means. We present a quantitative single-sarcomere model for isotonic SPOC in skeletal muscle with three basic ingredients: i), actin and myosin filaments initially in partial overlap, ii), stretch activation by length-dependent changes in the lattice spacing, and iii), viscoelastic passive tension. Modeling examples are given for slow-twitch and fast-twitch fibers, with periods of 10 s and 4 s respectively. Isotonic SPOC occurs in a narrow domain of parameter values, with small minimum and maximum values for actin-myosin affinity, a minimum amount of passive tension, and a maximum transient response rate that explains why SPOC is favored in slow-twitch fibers. The model also predicts the contractile, relaxed and SPOC phases as a function of phosphate and ADP levels. The single-sarcomere model can also be applied to a whole fiber under auxotonic and fixed-end conditions if the remaining sarcomeres are treated as a viscoelastic load. Here the model predicts an upper limit for the load stiffness that leads to SPOC; this limit lies above the equivalent loads expected from the rest of the fiber.

Bioequivalence study of a new oral topotecan formulation, relative to the current topotecan formulation, in patients with advanced solid tumors.

OBJECTIVE: The aims of this study were to investigate the bioequivalence of a new oral topotecan formulation (i.e., proposed commercial formulation) relative to the current oral formulation (formulation used in previous clinical trials), the effect of food on the absorption and disposition of the new oral topotecan and its safety and tolerability in patients with advanced solid tumors. PATIENTS AND METHODS: This was a multi-center, pharmacological Phase I, multiple-dose, randomized, open-label, cross-over bioequivalence study. In the bioequivalence part, 85 patients were randomized to receive either a 4 mg (4 x 1 mg) dose of the new or current formulation on Days 1 or 8. In the food-effect part, 23 patients received a 4 mg (4 x 1 mg) dose of the new formulation in a fasted and fed state. Total topotecan and topotecan lactone were determined and pharmacokinetic data were analyzed by non-compartmental method. RESULTS: Bioequivalence was demonstrated as the 90% confidence intervals of the ratio of the new to current formulation for both the area under the plasma concentration-time curve (AUC) and the maximal drug concentration (Cmax) for topotecan lactone were contained within the 0.8 - 1.25 boundary. The AUC and Cmax were similar in the fed and fasted state whilst food delayed the tmax for topotecan lactone and total topotecan. Safety data were collected on all subjects enrolled (n = 108) and were consistent with observations from previous studies of oral topotecan. All subjects experienced at least one adverse event, the majority of which were graded as mild to moderate in severity. CONCLUSION: The new oral topotecan formulation demonstrated bioequivalence to the current formulation and demonstrated it can be administered to patients with solid tumors in the fed or fasted state with similar systemic exposure.

Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets.

Lymphoma is a common tumor in ferrets, but anatomic distribution, histomorphology, immunophenotype, laboratory abnormalities, and response to chemotherapy are incompletely defined. In this study, lymphoma was diagnosed by histopathology of tumor tissue in 29 ferrets ranging in age from 0.8 to 8.5 years, including 12 males and 17 females. Tumors involved the viscera of the abdominal cavity (n = 11), thoracic cavity (n = 1), or abdominal and thoracic cavities (n = 7); the skin (n = 2); or the viscera of both body cavities plus other sites (n = 8). Microscopically, all tumors had diffuse architecture. Assessment by histomorphology and immunophenotype classified tumors as peripheral T-cell lymphoma (n = 17), anaplastic large T-cell lymphoma (n = 5), anaplastic large B-cell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 1), and Hodgkin-like lymphoma (n = 2). Cytologic evaluation of tumor tissue was diagnostic in 11 of 13 cases. Twenty-two of 27 ferrets had anemia, 2 had leukemia, and 5 were neutropenic. Common comorbid disorders were adrenal disease (n = 27) and insulinoma (n = 6). Tumors most frequently involved mesenteric lymph nodes, while enlargement of peripheral lymph nodes was uncommon (n = 3). Ferrets with Hodgkin-like lymphoma had massive enlargement of single lymph nodes. Mean survival of ferrets not immediately euthanized was 5.0 months (T-cell lymphoma) and 8.4 months (B-cell lymphoma). Ferrets treated with chemotherapy survived an average of 4.3 months (T-cell lymphoma, n = 9) or 8.8 months (B-cell lymphoma, n = 4). Results indicate that lymphomas in ferrets most commonly affect abdominal viscera, may be amenable to cytologic diagnosis, are frequently associated with anemia and, in some cases, may be chemosensitive, resulting in relatively long survival times.

Towards a unified theory of muscle contraction. I: foundations.

Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin-myosin-ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin-myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin-myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.

A new atomic force microscope force ramp technique using digital force feedback control reveals mechanically weak protein unfolding events.

We have developed a new force ramp modification of the atomic force microscope (AFM) which can control multiple unfolding events of a multi-modular protein using software-based digital force feedback control. With this feedback the force loading rate can be kept constant regardless the length of soft elastic linkage or number of unfolded polypeptide domains. An unfolding event is detected as a sudden drop in force, immediately after which the feedback control reduces the applied force to a low value of a few pN by lowering the force set point. Hence the remaining folded domains can relax and the subsequent force ramp is applied to relaxed protein domains identically in each case. We have applied this technique to determine the kinetic parameters x(u), which is the distance between the native state and transition state, and α(0), which is the unfolding rate constant at zero force, for the mechanical unfolding of a pentamer of I27 domains of titin. In each force ramp the unfolding probability depends on the number of folded domains remaining in the system and we had to take account of this effect in the analysis of unfolding force data. We obtained values of x(u) and α(0) to be 0.28 nm and 1.02 × 10(-3) s(-1), which are in good agreement with those obtained from conventional constant velocity experiments. This method reveals unfolding data at low forces that are not seen in constant velocity experiments and corrects for the change in stiffness that occurs with most mechanical systems throughout the unfolding process to allow constant force ramp experiments to be carried out. In addition, a mechanically weak structure was detected, which formed from the fully extended polypeptide chain during a force quench. This indicates that the new technique will allow studies of the folding kinetics of previously hidden, mechanically weak species.

A phase I and pharmacokinetic study of lapatinib in combination with infusional 5-fluorouracil, leucovorin and irinotecan.

BACKGROUND: This study determined the optimally tolerated regimen (OTR) of oral lapatinib administered in combination with infusional 5-fluorouracil (5-FU), leucovorin and irinotecan (FOLFIRI) and assessed the safety, tolerability and pharmacokinetics of the combination. PATIENTS AND METHODS: Twenty-five patients were enrolled; 12 patients were treated at three dose levels to determine OTR; then 13 patients were treated at OTR to evaluate the pharmacokinetics of the combination. RESULTS: The 2-weekly OTR comprised lapatinib 1250 mg/day with irinotecan 108 mg/m(2) (day 1) and leucovorin 200 mg/m(2), 5-FU bolus 240 mg/m(2) and 5-FU infusion 360 mg/m(2) (days 1 and 2); doses of 5-FU and irinotecan represent a 40% reduction in dose compared to conventional FOLFIRI. Dose-limiting toxicities were grade 3 diarrhoea and grade 4 neutropenia. Co-administration of lapatinib increased the area under the plasma concentration-time curve of SN-38, the active metabolite of irinotecan, by an average of 41%; no other pharmacokinetic interactions were observed. Of 19 patients evaluable for disease response assessment, four patients had partial response and nine patients had stable disease. CONCLUSION: The combination of lapatinib and FOLFIRI is safe and demonstrates clinical activity; the documented PK interaction can effectively be compensated by lowering the doses of 5-FU and irinotecan. This regime may be further tested in a phase II trial.

A nanoscale three-dimensional Monte Carlo simulation of electron-beam-induced deposition with gas dynamics.

A computer simulation was developed to simulate electron-beam-induced deposition (EBID). Simulated growth produced high-aspect-ratio, nanoscale pillar structures by simulating a stationary Gaussian electron beam. The simulator stores in memory the spatial and temporal coordinates of deposited atoms in addition to the type of electron, either primary (PE), back-scattered (BSE), or secondary (SE), that induced its deposition. The results provided in this paper apply to tungsten pillar growth by EBID on a tungsten substrate from WF(6) precursor, although the simulation may be applied to any substrate-precursor set. The details of the simulation are described including the Monte Carlo electron-solid interaction simulation used to generate scattered electron trajectories and SE generation, the probability of molecular dissociation of the precursor gas when an electron traverses the surface, and the gas dynamics which control the surface coverage of the WF(6) precursor on the substrate and pillar surface. In this paper, three specific studies are compared: the effects of beam energy, mass transport versus reaction-rate-limited growth, and the effects of surface diffusion on the EBID process.

Abdominal diameter index: a more powerful anthropometric measure for prevalent coronary heart disease risk in adult males.

AIM: The authors wished to compare the strength of association of several anthropometric measures of body size and fat distribution among themselves and in comparison with other known risk factors for prevalent coronary heart disease (CHD). METHODS: Prevalent CHD was assessed in 466 middle-aged, male, multiracial Triborough Bridge and Tunnel Authority officers in New York City by verified history, electrocardiogram or exercise stress test. Anthropometric measures included body mass index, waist, hip and thigh circumferences, waist-hip ratio, waist-thigh ratio, sagittal abdominal diameter and abdominal diameter index (sagittal abdominal diameter/thigh circumference). Results were compared with other CHD risk factors measured simultaneously (history of diabetes, smoking, blood pressure, lipid profile, apolipoproteins A and B, lipoprotein (a), homocysteine, fibrinogen, urinary microalbumin, serum vitamin E and ferritin) and a calculated 10-year CHD risk using a Framingham algorithm (10-year Framingham CHD risk). RESULTS: CHD was found in 29 individuals. Of the six anthropometric measures, abdominal diameter index gave the largest and most significant standardized odds ratio (OR) for CHD [1.80, 95% confidence interval (CI) 1.20, 2.71], equivalent to 10-year Framingham CHD risk. Men in the highest compared with the lowest tertile of abdominal diameter index had a univariate OR of 5.47 (95% CI 1.55, 19.28) which was the only anthropometric measure that remained significant after adjusting for 10-year Framingham CHD risk. CONCLUSIONS: For middle-aged American men, abdominal diameter index may be the most powerful anthropometric measure of risk for prevalent CHD.

Surface chemistry of enamel apatite during maturation in relation to pH: implications for protein removal and crystal growth.

Apatite crystal growth rate and morphology in mineralized tissues are considered to be controlled by surface interaction with extracellular matrix proteins. During enamel maturation where protein is finally removed from crystal surfaces to permit massive crystal growth, pH oscillates between approximately 5.8 and approximately 7.2. With this in mind, a study of enamel apatite surface chemistry in terms of local environmental pH was undertaken. Using atomic force microscopy adhesion force measurements were made between hydroxylated or carboxylated cantilever tips and maturation stage crystals between pH 2 and 10. Adhesion force increased from pH 10 to a maximum at pH 6.6 presumably due to increased hydrogen bonding due to replacement of surface cations (Na, Ca, Mg) with protons and/or protonation of phosphate per se. Below pH 6.6 adhesion force decreased and became very variable indicating that the surface had become unstable probably due to removal of fully protonated phosphate from the surface by adherence to the cantilever tip. Frictional force measurements also revealed 2-3, approximately 30 nm diameter high friction domains in bands across the crystal long axis. Their location mirrored the binding pattern of similarly sized amelogenin aggregates seen in vitro. The data suggests that specific protein binding sites may exist on crystal surfaces and may be released at lower pH by protonation which would lower cationic charge on both crystal surface and ionic charge on the protein. Instability of the crystal surface could also play a role.

Detection of Chlamydia pneumoniae in atherosclerotic tissue: a comparative study of PCR and immunocytochemistry.

The reported prevalence of Chlamydia pneumoniae in atherosclerotic tissue appears to depend on the detection system used. This introduces problems in determining the role of C. pneumoniae in atherosclerosis. This study analyses the sensitivity and performance of molecular diagnostic methods for the detection of C. pneumoniae and polymerase chain reaction (PCR) inhibitors in atheromatous tissue. Atherosclerotic tissue taken from 30 coronary endarterectomies, nine coronary arteries from explanted hearts, 16 carotid and two femoral endarterectomies are studied. Nested PCR (nPCR) assays targeting the PstI restriction fragment, the OmpA gene and the CRP operon of the chlamydial genome and immunocytochemistry (ICC) are used. Internal controls (IC) are constructed to co-amplify with the specific amplicons and identify the presence of inhibitor. The OmpA, PstI and CRP operon PCR assays had similar analytical sensitivities. However, the OmpA PCR was most affected by PCR inhibitors. Despite this, eight samples (14%) tested positive in the OmpA nPCR and no positives were found using the PstI or CRP operon nPCRs. Primary isolates of C. pneumoniae obtained from 12 patients with acute respiratory infection were positive in all three assays. Of the 48 specimens available for ICC, 33 (69%) were positive for chlamydial antigens. These included samples found positive by PCR. Dilution of samples to eliminate PCR inhibitors may have contributed to the discordant ICC and PCR results. The OmpA PCR, when used with an IC to identify samples with PCR inhibitors, is a reliable tool. However, the sensitivity of the ICC methods justifies their continued use.

Preservation of post-transplant lung function with aerosol cyclosporin.

Post-lung transplant use of aerosol cyclosporin (ACsA) is considered by examining the relationship between deposited aerosol dose and effect. In a sub-study of placebo controlled trials of ACsA as a rejection prophylaxis, 15 drug subjects received aerosol dose quantification tests to gage their ability to effectively deposit the nebulised drug in their transplanted lung(s). A total of seven placebo subjects received mock deposition tests. The deposited doses and mock doses were compared to changes in the forced expiratory volume in one second, at six time points during the 2-yr trial period (ACsA was started within 6 weeks post-transplant). Linear relationships were demonstrated between deposited dose and improvement in lung function in the drug subjects at all intervals. Mock dose data from placebo subjects did not demonstrate similar correlation. Based on these results, subjects were grouped by dose and compared. Subjects depositing > or = 5 mg of the drug in the periphery of their transplant(s) had improving pulmonary function on average. Low-dose and placebo subjects demonstrated declines, more A2-A4 rejection events in the latter portion of the trial, and more chronic rejection beyond the end of the trial. A dose-to-effect relationship is demonstrated for aerosol cyclosporin in terms of pulmonary function and biopsy proven rejection.