Integrated microscopy techniques for comprehensive pathology evaluation of an implantable left atrial pressure sensor.

The safety and efficacy of an implantable left atrial pressure (LAP) monitoring system is being evaluated in a clinical trial setting. Because the number of available specimens from the clinical trial for histopathology analysis is limited, it is beneficial to maximize the usage of each available specimen by relying on integrated microscopy techniques. The aim of this study is to demonstrate how a comprehensive pathology analysis of a single specimen may be reliably achieved using integrated microscopy techniques. Integrated microscopy techniques consisting of high-resolution gross digital photography followed by micro-computed tomography (micro-CT) scanning, low-vacuum scanning electron microscopy (LVSEM), and microground histology with special stains were applied to the same specimen. Integrated microscopy techniques were applied to eight human specimens. Micro-CT evaluation was beneficial for pinpointing the location and position of the device within the tissue, and for identifying any areas of interest or structural flaws that required additional examination. Usage of LVSEM was reliable in analyzing surface topography and cell type without destroying the integrity of the specimen. Following LVSEM, the specimen remained suitable for embedding in plastic and sectioning for light microscopy, using the positional data gathered from the micro-CT to intersect areas of interest in the slide. Finally, hematoxylin and eosin (H&E) and methylene blue staining was deployed on the slides with high-resolution results. The integration of multiple techniques on a single specimen maximized the usage of the limited number of available specimens from the clinical trial setting. Additionally, this integrated microscopic evaluation approach was found to have the added benefit of providing greater assurance of the derived conclusions because it was possible to cross-validate the results from multiple tests on the same specimen.

Coccidioidomycosis presenting as a heart base mass in two dogs.

Two atypical cases of canine coccidioidomycosis presenting as heart base masses are described. An echocardiogram performed in one of the two dogs revealed a large mass at the base of the heart and a computed tomography scan showed that the mass compressed the bronchi, left atrium, aorta and pulmonary arteries. A firm, white or pale yellow mass was found at the base of the heart at necropsy examination in both cases. Microscopical examination of the masses revealed severe, chronic, locally extensive granulomatous or pyogranulomatous inflammation with intralesional spherules consistent with Coccidioides spp. The diagnosis was further confirmed by immunohistochemistry and in-situ hybridization. Coccidioides spp. have been reported to cause pericarditis in dogs, but this is the first description of coccidioidomycosis mimicking a heart-based tumour in dogs.

Eight cases of feline cutaneous leishmaniasis in Texas.

Leishmaniasis is a zoonotic disease caused by intracellular Leishmania protozoa that are transmitted by sandflies. The disease occurs in 3 forms: cutaneous, mucocutaneous, and visceral. Cutaneous leishmaniasis has been reported in cats in Europe and South America and in 1 cat from Texas. Leishmania mexicana is endemic in Texas and has been reported to cause cutaneous lesions in humans. This article describes the pathology of 8 biopsy cases of feline cutaneous leishmaniasis presented to the Texas Veterinary Medical Diagnostic Laboratory over a 3.5-year period. The median age of the cats was 3 years; each was presented with nodular, ulcerative lesions on the pinnae and less commonly on the muzzle and periorbital skin. Histologically, the lesions were nodular to diffuse histiocytic dermatitis with numerous amastigotes (2-4 µm) within macrophages and occasionally within the interstitium. Organisms were often contained within round, clear, intracellular vacuoles. In areas of necrosis, organisms were also free within the interstitium. The overlying epidermis was hyperkeratotic, hyperplastic, and often ulcerated. The organisms were not argyrophilic (Gomori methenamine silver), reacted poorly with periodic acid-Schiff reagent, and were inconsistently basophilic with Giemsa. Although not readily visible histologically, kinetoplasts were evident in amastigotes in cytologic preparations. The lesions were similar to those described for cutaneous L. mexicana infection in humans. In 5 of the 8 cats, Leishmania mexicana DNA was amplified from paraffin-embedded tissue by polymerase chain reaction and sequenced.

Lack of an effect of vitamin E on lifespan of mice.

It has been speculated that ageing results from accumulation of damage to macromolecules, particularly DNA, owing to the action of oxidising free radicals. This possibility would predict that administration of anti-oxidants might prolong lifespan, but previous data on this prediction are conflicting. Three groups of mice were exposed throughout life, from the time of conception until death, to 20, 40 and 400 mg/kg of vitamin E in the diet. No effect on lifespan was observed and the median lifespans in the three groups were 804, 830 and 801 days, respectively. The design of the study also enabled an effect of parental age on lifespan of female progeny to be sought, but no effect was detected.

Rapid detection of the factor V Leiden (1691 G > A) and haemochromatosis (845 G > A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR.

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.

Immediate and reversible platelet inhibition after intravenous administration of a peptide glycoprotein IIb/IIIa inhibitor during percutaneous coronary intervention.

We studied the pharmacokinetic and pharmacodynamic properties of integrelin, a novel platelet glycoprotein IIb/IIIa receptor inhibitor, in patients undergoing elective percutaneous coronary intervention. Patients were randomized to placebo (n = 19) or to 1 of 4 integrelin dosing regimens (total n = 54) that were studied sequentially. All patients received aspirin and heparin. Patients were followed until discharge for the occurrence of adverse clinical events: death, myocardial infarction, coronary artery bypass surgery, repeat intervention, or recurrent ischemia. Bleeding was the primary safety end point. Frequent blood sampling was performed for adenosine diphosphate-induced platelet aggregations. Simplate bleeding times were performed. Adverse clinical events occurred less often in the integrelin-treated patients, although the overall numbers were too small to make a definitive statement as to clinical efficacy. There was no significant increase in serious bleeding among integrelin-treated patients. The 2 highest integrelin boluses (180 and 135 micrograms/kg) immediately (15 minutes after the bolus) provided > 80% inhibition of adenosine diphosphate-induced platelet aggregation in > 75% of treated patients. A constant integrelin infusion of 0.75 micrograms/kg/min maintained this marked antiplatelet effect, whereas an infusion of 0.50 micrograms/kg/min allowed gradual recovery of platelet function. Elective coronary intervention was performed safely and with no significant increase in serious bleeding events using integrelin with aspirin and heparin as an antithrombotic regimen. Integrelin provided rapid, intense, and persistent ex vivo platelet inhibition during coronary intervention. This new antiplatelet agent may be beneficial in reducing platelet-mediated ischemic complications of percutaneous coronary intervention.

A multifactorial intervention to reduce the risk of falling among elderly people living in the community.

BACKGROUND: Since falling is associated with serious morbidity among elderly people, we investigated whether the risk of falling could be reduced by modifying known risk factors. METHODS: We studied 301 men and women living in the community who were at least 70 years of age and who had at least one of the following risk factors for falling: postural hypotension; use of sedatives; use of at least four prescription medications; and impairment in arm or leg strength or range of motion, balance, ability to move safely from bed to chair or to the bathtub or toilet (transfer skills), or gait. These subjects were given either a combination of adjustment in their medications, behavioral instructions, and exercise programs aimed at modifying their risk factors (intervention group, 153 subjects) or usual health care plus social visits (control group, 148 subjects). RESULTS: During one year of follow-up, 35 percent of the intervention group fell, as compared with 47 percent of the control group (P = 0.04). The adjusted incidence-rate ratio for falling in the intervention group as compared with the control group was 0.69 (95 percent confidence interval, 0.52 to 0.90). Among the subjects who had a particular risk factor at base line, a smaller percentage of those in the intervention group than of those in the control group still had the risk factor at the time of reassessment, as follows: at least four prescription medications, 63 percent versus 86 percent, P = 0.009; balance impairment, 21 percent versus 46 percent, P = 0.001; impairment in toilet-transfer skills, 49 percent versus 65 percent, P = 0.05; and gait impairment, 45 percent versus 62 percent, P = 0.07. CONCLUSIONS: The multiple-risk-factor intervention strategy resulted in a significant reduction in the risk of falling among elderly persons in the community. In addition, the proportion of persons who had the targeted risk factors for falling was reduced in the intervention group, as compared with the control group. Thus, risk-factor modification may partially explain the reduction in the risk of falling.

Rural-urban child psychopathology in a Northeastern U.S. state: 1986-1989.

Parent and teacher symptom reports from two epidemiological surveys of 2,519 Connecticut children were used to study rural-urban differences in childhood psychopathology. Parents and teachers of girls in cities reported elevated total disturbance and social withdrawal. Parents of urban girls also reported higher rates of behavioral disturbance. For boys, urban excesses were primarily observed in emotional disturbance. Rural-urban variation was largely associated with economic and cultural differences between sites and not with urbanization per se. Findings suggest that certain assumptions about rural-urban differences in specific forms of psychopathology, such as delinquency, should be reevaluated.

Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction.

Gene rearrangement and monoclonality have been detected in normal cells and in lymphoproliferative disease by using the polymerase chain reaction and primers for the V and J regions of the Ig heavy chain gene or T-cell receptor gamma-chain gene. Using the Ig primers monoclonality was detected in 20 of 20 normal B-lymphocyte clones and in 39 of 52 cases of various types of B-lymphoproliferative disease, but not in 11 cases of T-lymphoproliferative disease. Using the T-cell receptor primers, monoclonality was detected in 186 of 192 normal T-lymphocyte clones, in 11 of 11 cases of T-lymphoproliferative disease, in 9 of 12 cases of B-acute lymphocytic leukemia, and in 1 of 21 cases of B-non-Hodgkin's lymphoma, but not in nine cases of B-chronic lymphocytic leukemia nor in 10 cases of myeloma. Monoclonality was detected in material obtained by lymph node aspiration in four of six additional cases of non-Hodgkin's lymphoma. It was not detected in 10 cases of acute myeloid leukemia nor in four cases of reactive lymphadenopathy. Detection of gene rearrangement by the polymerase chain reaction has a number of advantages over Southern blotting and is likely to become the initial diagnostic technique of choice to detect monoclonality.

Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to develop a simple technique for detecting monoclonality at the DNA level in B lymphocyte populations in formalin fixed, paraffin wax embedded material. Sections were dewaxed and dehydrated, and the DNA was extracted by boiling in water for 45 minutes. A semi-nested PCR was performed to amplify the V-D-J region of the immunoglobulin heavy chain gene. The product was electrophoresed and viewed under ultraviolet light after ethidium bromide staining. Specimens from 26 B cell lymphomas produced a monoclonal band in 24 cases and no amplification in two cases; monoclonality was specific for this disorder. Specimens from seven T cell lymphomas produced no amplification; specimens from nine reactive nodes produced a broad smear of polyclonal material; and specimens from 12 cases of carcinoma produced either no amplification or polyclonal material. As detection of monoclonality is strongly suggestive of neoplastic disease, this technique is likely to be of value in routine diagnosis, because of its speed, simplicity, and applicability to fixed, embedded material.

Monoclonality in B-lymphoproliferative disorders detected at the DNA level.

A new method was developed for detection of monoclonality at the DNA level in B-lymphoproliferative disease using the polymerase chain reaction and consensus primers for the V and J regions of the immunoglobulin gene. Monoclonality was detected in DNA from 15 of 15 clonal B-lymphoblastoid cell lines and from 19 of 23 cases of B-lymphocyte neoplasia, but not from any of 16 normal T-lymphocyte clones, 9 cases of T-lymphocyte neoplasia, 20 samples of polyclonal peripheral blood lymphocytes, and 8 cases of reactive lymphadenopathy. This method for detection of monoclonality is likely to be of routine value in diagnosis owing to its simplicity, speed, and versatility.

A patient registry for orthopedic surgery.

Identification of patient subpopulations for retrospective clinical studies, documentation of residents' clinical experience, and other administrative purposes can be difficult and time consuming. The problem of identification is exacerbated when a teaching program involves several hospitals or when the desired subpopulation is not adequately defined by standard diagnosis or procedure codes used by the institution. A useful patient registry system is reported here for the storage and retrieval of data on orthopedic patients treated by surgical residents at a major teaching hospital and its affiliates. The registry uses a simple, yet powerful encoding scheme to describe patient entries. In addition to a multidimensional encoded description based on SNOMED, the system supports the entry of free text to provide greater detail. This combination gives the patient registry both power and versatility.

Detection of the molecular abnormality in chronic myeloid leukemia by use of the polymerase chain reaction.

The bcr-abl translocation characteristic of chronic myeloid leukemia (CML) was detected by the polymerase chain reaction (PCR) modified to use mRNA as the starting material. Amplification of a sequence spanning the bcr-abl junction was obtained by using peripheral blood cells from all of 20 patients with classic CML, one patient with acute lymphoblastic leukemia probably secondary to CML, and two cell lines derived from patients with CML. The presence of bcr exon 3 in the mRNA was determined from the size of the amplified sequence; it was present in 14 and absent in seven patients. One leukemic cell per 1,000 nonleukemic cells could be readily detected, thus indicating the great sensitivity of the method. This technique is of routine value in CML both for diagnosis and for following the course of treatment.

Mutations in human lymphocytes studied by an HLA selection system.

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.

Activation of the food-derived mutagen 2-amino-3-methylimidazo[4, 5-f]quinoline by human-liver microsomes.

The ability of human-liver microsomes to metabolically activate the food-derived heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and the model mutagen, 2-aminofluorene (AF), has been investigated using Salmonella typhimurium TA98. In 6 subjects tested the number of revertants produced by 0.1 micrograms IQ per mg microsomal protein varied from 11, 830 +/- 320 to 42, 830 +/- 290 (mean +/- SD). With the same livers and a dose of 10 micrograms AF per plate the number of revertants varied from 15,770 +/- 1600 to 29,380 +/- 810 per mg microsomal protein. Metyrapone and alpha-naphthoflavone caused differential inhibition of the mutagenesis of both IQ and AF indicating the involvement of different forms of cytochrome P450 in the metabolic activation of these amines in human-liver microsomes. In presence of human-liver microsomes IQ produced no detectable increase in mutations at the hypoxanthine phosphoribosyl transferase locus in lymphocytes and caused no increase in micronuclei formation at realistic exposure levels.

Metabolism of 2-acetylaminofluorene in cultured human lymphocytes.

The capacity of lymphocytes from 23 human subjects to metabolize the model carcinogen 2-acetylaminofluorene (AAF) was assessed. These cells metabolized AAF to its 1-, 7- and N-hydroxylated metabolites. Alpha-naphthoflavone and metyrapone exhibited IC50's for all pathways of 0.02 microM and 1 mM, respectively. These data indicate that the same form of cytochrome P450 or forms with similar IC50's are mediating these reactions. No significant difference was observed between lymphocytes from smokers versus non-smokers in the N-hydroxylation of AAF, the initial step in the metabolic activation of this carcinogen. However, lymphocytes from smokers were faster detoxifiers of AAF as seen in their increased capacity to 1- and 7-hydroxylate AAF compared to lymphocytes from non-smokers.

Mutation rate of normal and malignant human lymphocytes.

The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells.

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.

Methods for study of mutations and mutagenesis in human lymphocytes.

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.

Mutation frequency in human lymphocytes increases with age.

Several theories of ageing predict that somatic mutations should increase with age. This prediction was tested for human lymphocytes using a recently developed clonal technique for enumeration of mutations, and an increase of 1.6% per year in mutations with age was detected.