Automatic correction of continuum background in laser-induced breakdown and Raman spectrometry.

The goal of this work was the development and evaluation of an algorithm for the approximation and automatic subtraction of continuum backgrounds in laser-induced breakdown and Raman spectra. The background correction algorithm was applied to simple and complex spectra and its effect on identification accuracy was studied. Linear correlation was used for the identification of plastic samples using both laser-induced breakdown and Raman spectra. For both techniques, the algorithm successfully eliminated continuum background without compromising spectral integrity. A significant improvement in the percentage of correct plastic identifications was observed for Raman spectra. The approach should be applicable to a wide range of background correction problems in atomic and molecular spectroscopy.

Solutions to health care waste: life-cycle thinking and "green" purchasing.

Health care waste treatment is linked to bioaccumulative toxic substances, such as mercury and dioxins, which suggests the need for a new approach to product selection. To address environmental issues proactively, all stages of the product life cycle should be considered during material selection. The purchasing mechanism is a promising channel for action that can be used to promote the use of environmentally preferable products in the health care industry; health care facilities can improve environmental performance and still decrease costs. Tools that focus on environmentally preferable purchasing are now emerging for the health care industry. These tools can help hospitals select products that create the least amount of environmental pollution. Environmental performance should be incorporated into the evolving definition of quality for health care.

Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load.

Epstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.

Accumulation of p53 in infectious mononucleosis tissues.

Epstein-Barr virus (EBV) infects lymphocytes, where it persists indefinitely for the life of the host; whether the virus interacts with p53 to maintain itself in these cells is unknown. Lymphoid biopsy samples from 10 patients with infectious mononucleosis (IM) were examined for expression of p53 by immunohistochemistry. Accumulation of p53 was detected in all 10 cases, primarily in large lymphocytes of the expanded paracortex. The presence of EBV was confirmed in all 10 cases by EBER1 (EBV-encoded RNA) in situ hybridization, whereas 11 non-IM control samples lacked significant EBER1 and did not express p53 in paracortical lymphocytes. Interestingly, EBV infection alone does not cause accumulation of intracellular p53, because many more cells expressed EBER1 than p53 in the IM tissues. To determine whether p53 was confined to the subset of infected cells in which viral replication was occurring, BZLF1 immunostains were performed. Viral BZLF1 was detected in 8 of 10 IM tissues; however, the paucity and small size of the BZLF1-expressing lymphocytes suggests that they are not the same cells overexpressing p53. To further examine the relationship between p53 and EBV gene expression, the tissues were studied for latent membrane protein 1 (LMP1) expression by immunohistochemistry. Viral LMP1 was observed in the large paracortical lymphocytes of all 10 cases of IM, indicating co-localization of p53 and LMP1 in these cells. Our findings confirm that p53 overexpression is not specific for nodal malignancy and that p53 accumulation is characteristic of IM. Because p53 was not coexpressed in the same cells as BZLF1, it appears that BZLF1 is not directly responsible for p53 accumulation. Nevertheless, co-localization of p53 and LMP1 in activated-appearing lymphocytes suggests that EBV infection is responsible for p53 accumulation. HUM PATHOL 31:1397-1403.

Loss of p16/CDKN2A tumor suppressor protein in gastric adenocarcinoma is associated with Epstein-Barr virus and anatomic location in the body of the stomach.

Gastric adenocarcinomas (n = 125) were analyzed by immunohistochemistry for the presence of p16, the CDKN2A gene product. This protein was lost in 31 of 125 cases (25%), and loss was associated with location of the tumor in the body of the stomach (P = .001). Loss of p16 was also associated with the presence of Epstein-Barr virus (EBV) in tumor cells as determined by in situ hybridization (P = .022). This effect may relate to anatomic site, because EBV-associated tumors originate more frequently in the body of the stomach. When p16 status was evaluated for ethnic origin of the patient (non-Hispanic white, Hispanic, or black), a strong trend (P = .057) was found for African-American patients to have fewer p16-negative tumors than other patients. This also may relate to anatomic location, because fewer tumors from black patients arose in the body of the stomach (P = .022). No significant associations were detected between p16 status and histological subtype (intestinal v diffuse), the presence of microsatellite instability, grade or stage of the tumor, or age, gender, or survival of the patient. In conclusion, p16 loss is quite common in gastric adenocarcinoma, and such loss is more common in EBV-infected tumors arising in the body of the stomach.

Absence of Epstein-Barr virus in lymphomatoid papulosis. An immunohistochemical and in situ hybridization study.

BACKGROUND AND DESIGN: Lymphomatoid papulosis (LyP) and cutaneous Hodgkin's disease share many clinical, histopathologic, and immunohistochemical features. Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several lymphoid malignancies, including Hodgkin's disease. Given the similarities between LyP and Hodgkin's disease, we asked if EBV could be detected in lesions of LyP. We examined 31 specimens of LyP that were obtained from 24 patients for evidence of EBV by in situ hybridization to EBER1 transcripts and for immunohistochemistry of viral latent membrane protein 1 (LMP1). RESULTS: In no instance there was there any evidence of EBV gene products by either in situ hybridization or immunohistochemistry. CONCLUSIONS: The absence of EBV in LyP suggests that this virus is not operative in the pathogenesis of LyP. Furthermore, it suggests that LyP and Hodgkin's disease may not share the same molecular mechanisms despite their phenotypic similarities.

Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation.

Ninety-five cases of adenocarcinoma of the stomach were evaluated for the presence of Epstein-Barr virus (EBV) using a sensitive in situ hybridization assay targeting Epstein-Barr virus-encoded RNA 1 (EBER1) transcripts. EBER1 was detected in 11 of 95 (12%) of cases. When present, the virus was localized to malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in normal-appearing gastric epithelium or intestinal metaplasia. The EBV DNA was monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant transformation. The presence of EBV was strongly associated with increased numbers of tumor-infiltrating T lymphocytes; however, EBV was not associated with prolonged survival. Neither p53 nor bcl-2 were consistently detected in the EBV-associated tumors. Specifically, 6 of 11 EBV-positive carcinomas had accumulation of p53 protein by immunohistochemical analysis, which was similar to the prevalence of p53 accumulation in EBV-negative specimens and suggests that EBV infection does not substitute for p53 mutations during tumorigenesis. The bcl-2 oncoprotein was expressed in a third of the carcinoma specimens tested, but bcl-2 expression did not correlate with the presence of EBV or with expression of EBV latent membrane protein 1. In conclusion, EBV infection appears to precede malignant transformation in a significant fraction of gastric carcinomas, but neither bcl-2 expression nor p53 accumulation appear to be consistently associated with the presence of the virus.

Epstein-Barr virus is detected in undifferentiated nasopharyngeal carcinoma but not in lymphoepithelioma-like carcinoma of the urinary bladder.

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.

Lymphomas of the oral soft tissues are not preferentially associated with latent or replicative Epstein-Barr virus.

OBJECTIVES: Epstein-Barr virus is periodically shed in the saliva of persons infected by the virus. Epstein-Barr virus has been implicated in the pathogenesis of certain subtypes of lymphoma, particularly high-grade lymphomas. Because high-grade subtypes represent the majority of lymphomas that arise in oral soft tissues, we hypothesized that Epstein-Barr virus might be preferentially associated with oral lymphomas. STUDY DESIGN: A series of 34 oral lymphomas were diagnosed according to the revised European-American classification scheme. They were examined for the presence of latent Epstein-Barr virus by EBER1 in situ hybridization and for expression of the Epstein-Barr virus replicative protein, BZLF1, by immunohistochemistry. RESULTS: Epstein-Barr virus EBER1 transcripts were detected in 11 of 31 oral lymphomas including 7 of 10 AIDS-related lymphomas and only 4 of 21 lymphomas that occurred in nonimmunocompromised persons. The Epstein-Barr virus-containing lymphomas were all high-grade histologic subtypes, that is, diffuse large cell, immunoblastic, or Burkitt's lymphomas. In contrast, Epstein-Barr virus was not detected in any of five low-grade oral lymphomas. In the single case of T-cell lymphoma in this study, EBER1 was expressed in the tumor cells. A switch from viral latency to replication, as measured by EBV BZLF1 expression, was identified in rare lymphoma cells in only four cases. This rate of viral replication was not higher than what has been reported in lymphomas arising at other anatomic sites. Although one of our lymphomas arose at a site of previous oral hairy leukoplakia, there was no other evidence that Epstein-Barr virus replication predisposed to development or persistence of oral lymphomas. CONCLUSIONS: These data suggest that even though Epstein-Barr virus is frequently found in oral secretions, neither latent nor replicative Epstein-Barr virus is present more commonly in oral lymphomas than in lymphomas arising in other anatomic sites, when controlling for immunodeficiency status.

Diagnosis of Epstein-Barr virus associated nasopharyngeal carcinoma using fine-needle aspiration biopsy and molecular diagnostics.

Molecular technology is being utilized increasingly for diagnostic purposes by practicing pathologists. Techniques such as Southern blot, in situ hybridization, and polymerase chain reaction have recently been introduced to the clinical laboratory setting. We describe a case of nasopharyngeal carcinoma that highlights the potential utility of DNA technology to secure an accurate diagnosis of a fine-needle aspiration biopsy. In this patient, cytologic examination of a cervical lymph node aspirate strongly suggested the possibility of a nasopharyngeal carcinoma. Needle aspirate material was submitted for molecular genetic detection of the Epstein-Barr virus (EBV) genome. Nine micrograms of DNA were isolated, and the presence of clonal EBV DNA was detected by the Southern blot technique. The presence of clonal EBV supported the cytologic diagnosis of nasopharyngeal carcinoma. Subsequent biopsy of a nasopharyngeal mass revealed undifferentiated carcinoma, and in situ hybridization revealed that EBV was restricted to the malignant epithelial cells. This case illustrates how molecular technology can provide new information that is useful in diagnostic cytopathology.

Absence of a reservoir of Epstein-Barr virus (EBV) in normal tongue epithelium.

We examined human tongue epithelium and serum samples at autopsy for evidence of latent Epstein-Barr virus (EBV) infection. Although clinical serology revealed anti-EBV antibodies in most sera indicating past EBV infection, we found no Epstein-Barr nuclear antigen (EBNA)-coding sequences in tongue tissue by polymerase chain reaction (PCR), or Epstein-Barr-encoded RNA (EBER1) by in situ hybridization. Tongue epithelium does not appear to be a natural reservoir for latent EBV in immunocompetent hosts.

Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Sternberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity.

One hundred twenty-five cases of Hodgkin's disease from the United States (79), Mexico City (31), and Costa Rica (15) were analyzed for the presence of Epstein-Barr virus (EBV) by in situ hybridization to EBER1 transcripts. EBV was more frequently detected in the Reed-Sternberg (RS) cells of mixed cellularity Hodgkin's disease (37 of 48 [77%]) compared with the nodular sclerosis subtype (19 of 71 [27%], P < .001). The presence of EBV was also associated with Hispanic ethnicity (P < .001). In a multivariate analysis, patient age, gender, and geographic location were less predictive of EBV positivity than were mixed cellularity histology (odds ratio = 8.3) and Hispanic ethnicity (odds ratio = 4.3). Southern blot analysis of EBV terminal repeat fragments using the Xho1a probe showed that the viral DNA was monoclonal in 17 of 17 cases having EBER1-positive RS cells. By comparison, EBV DNA was not detected by Southern analysis in 20 cases lacking EBER1 in RS cells, even when occasional background lymphocytes expressed EBER1. Because clonal viral DNA was so readily detected in EBER1-positive cases, the EBV genome is probably amplified at least 50-fold in the infected RS cells. Monoclonality of EBV DNA implies that the RS cells were infected before malignant transformation.

gamma-Aminobutyric acid localization and function as modulator of cholinergic neurotransmission in rat antral mucosal/submucosal fragments.

gamma-Aminobutyric acid, a neurotransmitter in the central nervous system, has been shown to be present in and synthesized and secreted by rodent and feline myenteric plexus neurons. The aims of the present studies were to measure gamma-aminobutyric acid concentrations and synthesis and to establish cellular localization and uptake of gamma-aminobutyric acid by immunocytochemistry and autoradiography, respectively, within mucosal and submucosal tissues of the rat antrum. Direct demonstration of [3H]gamma-aminobutyric acid release and the effects of exogenous gamma-aminobutyric acid and muscimol, a GABA alpha agonist, on [3H]acetylcholine release from antral mucosal/submucosal fragments were examined in perifusion experiments. gamma-Aminobutyric acid content and synthesis, as reflected by glutamic acid decarboxylase activity, were present within antral mucosa at levels two to three times that of the body and muscular layers of both the gastric body and antrum. gamma-Aminobutyric acid was identified immunocytochemically, principally in mucosal epithelial cells of the antrum. Exogenous gamma-aminobutyric acid and muscimol were capable of stimulating acetylcholine release through a GABA alpha receptor-mediated mechanism that was abolished by tetrodotoxin. These results indicate that gamma-aminobutyric acid is present in and taken up by epithelial cells of the gastric antrum and that gamma-aminobutyric acid is capable of being synthesized by antral mucosal/submucosal tissues. Furthermore, these studies suggest that a peripheral gamma-aminobutyric acid mechanism that may modulate cholinergic neurotransmission and endocrine cell function exists within the antrum.

Hyperthermic effects on viability and growth kinetics of human lymphoblastoid cells.

During clinical hyperthermia, various blood elements may be exposed to elevated temperatures. The effect of heat on human lymphocyte viability and human lymphoblastoid cell viability and growth was therefore measured. In the viability studies, cells were heated for different times and temperatures and stained with fluorescein diacetate either immediately of at various times after treatment; dye uptake was then analysed using fluorescence microscopy. There was no significant decrease in lymphocyte viability when assayed at 0 and 24 h after heating at 42-43 degrees C for varying times. Similarly, when proliferating lymphoblastoid cells were heated at 42-43 degrees C, there was no decrease measured in viability immediately after heating. However, in contrast to the lymphocyte results, a progressive decrease of lymphoblastoid cell viability was observed with increasing time after treatment. A nadir in viability was observed 48-72 h after heating, followed by a subsequent apparent recovery. This recovery showed a correlation with cell growth, as well as lysis of non-viable cells. The cell population doubling time was also lengthened, with longer doubling times observed for more severe heat treatments.

Proflavin and microwave radiation: absence of a mutagenic interaction.

The potential ability of radiofrequency electromagnetic radiation (RFR) in the microwave range to induce mutagenesis, chromosomal aberrations, and sister chromatid exchanges in mammalian cells is being explored in our laboratories. In addition, we have also been examining the ability of simultaneous exposure to RFR and chemical mutagens to alter the genotoxic damage induced by chemical mutagens acting alone. We have performed experiments to determine whether there is an interaction between 2.45-GHz, pulsed-wave, RFR and proflavin, a DNA-intercalating drug. The endpoint studied was forward mutation at the thymidine kinase locus in L5178Y mouse leukemic cells. Any effect on the size distribution of the resulting colonies of mutated cells was also examined. The exposures were performed at net forward powers of 500 or 600 W, resulting in a specific absorption rate (SAR) of approximately 40 W/kg. The culture-medium temperature reached a 3 degrees C maximal increase during the 4-h exposure; appropriate 37 degrees C and convection-heating temperature controls (TC) were performed. In no case was there any indication of a statistically significant increase in the induced mutant frequency due to the simultaneous exposure to RFR and proflavin, as compared with the proflavin exposures alone. There was also no indication of any change in the colony-size distribution of the resulting mutant colonies, neither, and there was no evidence in these experiments of any mutagenic action by the RFR exposure alone.

137Cs dosimetry table for asymmetric source.

A common 137Cs brachytherapy source has a 2-cm physical length and 1.38-cm active length. The active length is not symmetric with respect to the source center because one source end contains an eyelet. Current dose rate tables assume a symmetric source loading with respect to the source center. A computer program was written to calculate an asymmetric distribution using the manufacturers' source specifications. Corrections were made for attenuation and obliquity through the source materials. Dose rate values in cGy/h for a 137Cs source equivalent to 1 mg of 226Ra are shown as a function of the radius from the source center and angle from the source end containing the eyelet. Dose rate values in the table were confirmed by ferrous sulfate measurements using small volumes. The table values agree with published values at points more distant than a few cm and lying at an angle such that the asymmetry of the source loading has its minimum influence.

Absence of mutagenic interaction between microwaves and mitomycin C in mammalian cells.

Evidence in the literature from in vitro and in vivo studies as to whether or not radiofrequency radiation (RFR) in the microwave range is mutagenic is predominantly negative, with some positive reports. No evidence is available as to whether RFR will alter the mutagenic activity of genotoxic chemicals during a simultaneous exposure, a likely real-life situation. Two hypotheses have been proposed: a) that RFR by itself can cause mutations in a mammalian cell in vitro assay system; and b) that a simultaneous exposure to RFR during a chemical treatment of the cells with a known genotoxic agent, mitomycin C (MMC), will alter the extent of mutagenesis induced by the treatment of the cells by the chemical alone. These studies were performed using the forward mutation assay at the thymidine kinase locus in L5178Y mouse leukemic cells. The pulsed wave RFR was broadcast from an antenna horn at a frequency of 2.45 GHz. The power density was 48.8 mW/cm2 and the measured specific absorption rate (SAR) in this system was 30 W/kg (600 W forward power), which is well above current safety guidelines. The conclusions from five different experiments, employing three different concentrations of MMC, were that a) RFR exposure alone, at moderate power levels which resulted in a temperature increase in the cell culture medium of less than 3 degrees C, is not mutagenic; and b) when cells are simultaneously treated with MMC and RFR at these same moderate power levels, the RFR does not affect either the inhibition of cell growth or the extent of mutagenesis resulting from the treatment with the chemical MMC alone.

Dosimetry considerations in far field microwave exposure of mammalian cells.

A circulating water bath exposure system has been designed for in vitro radiofrequency radiation (RFR) exposure studies in the 915 to 2450 MHz range. A Styrofoam float, in which 10 T-25 plastic tissue culture flasks are embedded, is rotated at approximately 20 rpm in a Plexiglas water bath at a distance beneath a rectangular horn. The continuous circular rotation of the flasks is designed to "average out" the heterogeneity present in stationary flask exposures. The rotation also serves to prevent the establishment of chemical gradients in the medium within the flasks. Several factors have been demonstrated to affect the specific absorption rate (SAR) measured in the medium in the exposed flasks. These factors include: 1) the position of the exposure flasks relative to the long axis of the antenna horn; 2) whether the flasks are exposed while stationary or in rotation; 3) the volume of the medium contained in the flask; and 4) the depth in the medium in the flask at which temperatures for SAR calculation are measured. The presence of cells in the exposure flask (as attached monolayer or cell suspension) did not result in an SAR different from that measured in the same volume of medium without cells present.

New clinical roles for pharmacists: a study of role expansion.

This study assessed the legitimacy of expanded roles for pharmacists with different status audiences. Pharmacy is a profession in transition and is characterized by considerable ambiguity and uncertainty concerning its status as a health care profession. Significant changes have occurred within the profession of pharmacy in the past few decades which have led to loss of function, social power and status. The response of the profession has been a movement toward a patient-oriented, clinical role for pharmacists. Hypotheses concerning level of support for expanded roles were derived from two conflict-based models of professionalization: a power model which focuses on conflict between professions and the central role of power in defining occupational territory; and a process model which focuses on conflict of interest and diversity within a profession and the development of 'segments' which struggle for control of a profession's direction. Data were collected by self-administered questionnaires sent to California pharmacists, physicians and nurses. Respondents were asked to indicate level of support for 20 role activities for pharmacists working in two practice settings (community and hospital). Pharmacy faculty were the most supportive of the clinical role activities, followed by practicing pharmacists, nurses and physicians. Physicians and nurses were more antagonistic toward clinical activities in the community than hospital practice setting, and were most antagonistic toward role activities which require independent judgement or autonomous action relevant to patient care on the part of the pharmacist. Differences were also noted in support for clinical role activities within the pharmacists' group. The effect of experience in working with a clinical pharmacist on support for clinical role activities is also discussed.

Absence of endotoxin fever but not prostaglandin E2 fever in the Brattleboro rat.

Changes in colonic temperature following intracerebroventricular injection (icv) of bacterial endotoxin or prostaglandin E2 (PGE2) were measured in Long-Evans (LE) and Brattleboro (DI) rats. Indwelling cannulas were implanted into the brains of rats for subsequent microinjection into a lateral cerebral ventricle. Microinjection of 1 microgram of bacterial endotoxin into a lateral cerebral ventricle produced a fever in the LE rat but not in the DI rat. Daily injections of 1 microgram of endotoxin icv in the DI rat did not result in a fever. Intraperitoneal injections of 50 micrograms of bacterial endotoxin resulted in a fever in the LE rat, but the DI rat showed no such response. Both groups of animals did produce a fever in response to icv administration of 200 ng of PGE2. The lack of arginine vasopressin in the DI rat may be related to the animal's failure to show a febrile response to endotoxin.