Acute effects of prostaglandin E1 and E2 on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.

Telepathology overview: from concept to implementation.

Telepathology is the practice of pathology at a distance by using video imaging and telecommunications. Significant progress has been made in telepathology. To date, 12 classes of telepathology systems have been engineered. Rapid and ultrarapid virtual slide processors may further expand the range of telepathology applications. Next-generation digital imaging light microscopes, such as miniaturized microscope arrays (MMA), may make virtual slide processing a routine laboratory tool. Diagnostic accuracy of telepathology is comparable with that of conventional light microscopy for most diagnoses. Current telepathology applications include intraoperative frozen sections services, routine surgical pathology services, second opinions, and subspecialty consultations. Three telepathology practice models are discussed: the subspecialty practice (SSP) model; the case triage practice (CTP) model; and the virtual group practice (VGP) model. Human factors influence performance with telepathology. Experience with 500 telepathology cases from multiple organs significantly reduces the video viewing time per case (P < .01). Many technology innovations can be represented as S-curves. After long incubation periods, technology use and/or efficiency may accelerate. Telepathology appears to be following an S-curve for a technical innovation.

Combined robotic and nonrobotic telepathology as an integral service component of a geographically dispersed laboratory network.

To achieve real-time connectivity between its 8 hopital-based laboratories, Veterans Integrated Service Network (VISN) 12, headquartered in Chicago, IL, has implemented a hybrid dynamic store-and-forward (HDSF) telepathology network that extends across portions of 3 states. The majority of diagnostic telepathology functions are provided to the 3 hospitals (Iron Mountain, MI; Tomah, WI; and North Chicago, IL), which lack on-site pathologists and are serviced by the 4 pathologists located in Milwaukee, WI. In surgical pathology, routine primary diagnosis, frozen section diagnosis, and clinical consultation are provided with telepathology. In addition, autopsy and specialty clinical conferences are frequently performed by using telepathology. Telepathology has been applied to a variety of areas within clinical pathology as well, including protein electrophoresis, immunoelectrophoresis, peripheral blood smears, body fluids, microbiology, and distance learning. Implementation of telepathology has allowed VISN 12 to reach the goal of providing a single standard of accurate and timely pathology service, even at small sites that lack an on-site pathologist.

Telepathology networking in VISN-12 of the Veterans Health Administration.

The Veterans Integrated Service Network (VISN)-12, headquartered in Chicago, has implemented a telepathology network between the eight VISN-12 hospital laboratories and Loyola University Medical School linked by an economical, high-speed wide-area network (WAN). Implementation of the WAN has reduced monthly telecommunications costs in VISN-12 by approximately 67%. In addition to telepathology, the WAN enables real-time teleradiology (general, computer tomography, and ultrasound), telefluoroscopy, telenuclear medicine imaging, telepsychiatry, and other forms of teleconsultation. Current applications of telepathology in VISN-12 include: primary diagnosis and consultation in surgical pathology, interpretation of serum protein electrophoresis and immunofixation gels, provision of support for consolidated microbiology laboratories, review of problematic peripheral blood smears, and distance learning. We have learned a variety of lessons from telepathology. The enthusiasm and technical skill of providers are essential for success. As well, frequent communication and rapid technical support are necessary. Finally, in a supportive environment, telepathology is a tool that can help bring together clinical laboratories with shared missions and goals.

Cost minimization analysis of telepathology.

Telepathology is gaining acceptance as a mode of providing pathology services to remote sites, but its economic feasibility is unknown. A dynamic robotic telepathology service between the Veterans Affairs Medical Center, Iron Mountain, MI, and the Veterans Affairs Medical Center, Milwaukee, WI, provides diagnostic services for routine and frozen section surgical pathology cases at Iron Mountain. We conducted a cost minimization analysis of this service by building a model to compare telepathology and on-site pathology in Iron Mountain and a courier method of transporting specimens from Iron Mountain to Milwaukee for diagnosis. Base case analysis showed the courier method to be the most economic; telepathology was less costly than on-site pathology. If the costs of telepathology equipment and telecommunication are lowered to reflect current cost, then telepathology becomes the favored option. Telepathology can be an economic mode of providing pathology services to a remote site.

Identification of a novel penicillin-binding protein from Helicobacter pylori.

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.

Unsedated transnasal EGD: an alternative approach to conventional esophagogastroduodenoscopy for documenting Helicobacter pylori eradication.

BACKGROUND: The aim of this study was to assess the yield of antral biopsies performed via unsedated transnasal esophagogastroduodenoscopy, a technique that does not require conscious sedation with its concomitant costs and complications, for documentation of Helicobacter pylori eradication. METHODS: Nineteen patients who were previously CLO test positive on conventional esophagogastroduodenoscopy and subsequently treated for H pylori infection were enrolled. The subjects had not received antibiotic therapy in the prior month and had no prior gastric surgery. By using a GIF-N30 fiberoptic endoscope and a tiny cup biopsy forceps (1.8 mm diameter), unsedated transnasal endoscopy was performed and antral biopsy specimens were taken for a CLO test, histologic analysis (Dieterle stain), and tissue culture. On the same day, the subjects underwent a carbon 13-labeled area urea breath test. All subjects completed a visual analog scale, rating the acceptability of the unsedated transnasal examination and the previous sedated conventional esophagogastroduodenoscopy. RESULTS: There was no statistically significant difference between the results of the CLO tests (5/19 positive) versus the 13C-urea breath test (4/19 positive) (p = 0.96), the CLO tests versus histologic findings (5/19 positive) (p = 0.71), or the 13C-urea breath test versus histologic findings (p = 0.96). All tissue culture results were negative. The overall acceptability of unsedated transnasal esophagogastroduodenoscopy was similar to that of sedated conventional esophagogastroduodenoscopy. CONCLUSION: Unsedated transnasal esophagogastroduodenoscopy, a technique that eliminates the costs and complications associated with conscious sedation, is a feasible and accurate alternative to conventional esophagogastroduodenoscopy when documentation of H pylori eradication and confirmation of gastric ulcer healing are both indicated.

Routine surgical telepathology in the Department of Veterans Affairs: experience-related improvements in pathologist performance in 2200 cases.

OBJECTIVE: To determine whether diagnostic concordance, case deferral rate, and/or time required to review slides changed significantly as telepathologists gained additional experience using a hybrid dynamic/store-and-forward (HDSF) telepathology (TP) system on the 2000 cases following an initial 200 consecutive surgical cases, previously reported. MATERIALS AND METHODS: Gross surgical pathology specimens were prepared by specially trained personnel in Iron Mountain, Michigan. For TP, glass slides were placed on the stage of a robotic microscope at the Iron Mountain VAMC (remote site); control of the motorized microscope was then transferred to a pathologist located 220 miles away at the Milwaukee, Wisconsin, VAMC (host site). For each case, a telepathologist had the option of either rendering a diagnosis or deferring the case for later analysis by conventional light microscopy (LM). After the slides were read by TP and a surgical pathology report had been generated (for nondeferred cases), the slides were transported to Milwaukee, where they were reexamined by the same pathologist, now using LM. When there was disagreement between the TP and LM diagnosis, a supplemental or revised report was issued, and the referring physician was notified by telephone immediately. All supplemental and revised reports were reviewed by a third pathologist in the group. The slides were then reviewed by the pathology group practice or, when there was no consensus, by the Armed Forces Institute of Pathology to establish a "truth" diagnosis. To determine changes in telepathologist performance with experience after the initial start-up of the service, their performance in handling 10 consecutive sets of 200 surgical pathology cases was analyzed. RESULTS: Concordance rates for clinically significant TP and LM diagnoses were high for all 10 sets, ranging from 99% to 100%. Comparing the first set (Cases 201-400) with the last set (Cases 2001-2200), viewing times per case were reduced from 10.26 min to 3. 58 min. Viewing times per slide were reduced from 3.44 min to 1.13 min per slide, comparing the first and last sets. Case turnaround times (TAT) decreased from 2.46 days to < or =1.5 days. CONCLUSION: Thes results demonstrate that improvements in TP services occur over time as the result of additional experience using the TP system. The high diagnostic concordance and low rate of case deferral lend additional support to the proposal that a host-site pathologist using HDSF TP can substitute effectively for an on-site pathologist as a service provider.

Helicobacter pylori containing only cytoplasmic urease is susceptible to acid.

Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 microM), a potent, but poorly diffusible urease inhibitor. H. pylori with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly, Escherichia coli SE5000 expressing H. pylori urease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type H. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pylori in acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria ("altruistic autolysis"), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.

Telemicrobiology: feasibility study.

BACKGROUND: Rural hospitals generally lack staffing with infectious disease specialists or pathologists. Without on-site pathologists, the range of microbiology services offered by clinical laboratories may be limited as well. OBJECTIVE: To study the feasibility of using static-image telepathology to evaluate Gram stains of microbiologic preparations. MATERIALS AND METHODS: In this retrospective feasibility study, three pathologists evaluated Gram stains of slides from 50 cases by two viewing modalities: static-image telepathology and conventional light microscopy. Digital video images of slides were captured at two magnifications (using 40x and 100x objective lenses) at 1024 x 768 x 24-bit color and transmitted over standard telephone lines at 14,400 kbps. Pathology reports and culture results served as "truth diagnoses." Categories of interpretations were correct, minor discrepancy, or major discrepancy with regard to the implications for patient care. RESULTS: The diagnostic accuracy of video image readings and conventional light microscopy readings were nearly identical, with no statistically significant differences in the performances of specialty and nonspecialty pathologists (P > 0.05). The mean accuracies of readings of the video images and light microscopy images were 95.3% and 95.4%, respectively. Taking into account the time required by a referring pathologist to capture video digital images, telemicrobiology was somewhat less efficient than conventional light microscopy. CONCLUSIONS: Pathologists can accurately evaluate digital video images of preselected fields on Gram-stained slides. In clinical practice, however, a limiting factor may be the availability of local personnel qualified to select the microscopic fields for evaluation by telepathologists. The adequacy of the video images suggests that telepathology may also be used for remote supervision of quality assurance programs in microbiology laboratories, as well as for remote proficiency training of laboratory personnel.

Structure, function and localization of Helicobacter pylori urease.

Helicobacter pylori is the causative agent of most cases of gastritis. Once acquired, H. pylori establishes chronic persistent infection; it is this long-term infection that, is a subset of patients, leads to gastric or duodenal ulcer, gastric cancer or gastric MALT lymphoma. All fresh isolates of H. pylori express significant urease activity, which is essential to survival and pathogenesis of the bacterium. A significant fraction of urease is associated with the surface of H. pylori both in vivo and in vitro. Surface-associated urease is essential for H. pylori to resist exposure to acid in the presence of urea. The mechanism whereby urease becomes associated with the surface of H. pylori is unique. This process, which we term "altruistic autolysis," involves release of urease (and other cytoplasmic proteins) by genetically programmed autolysis with subsequent adsorption of the released urease onto the surface of neighboring intact bacteria. To our knowledge, this is the first evidence of essential communal behavior in pathogenic bacteria; such behavior is crucial to understanding the pathogenesis of H. pylori.

Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori has been found in the stomachs of humans in all parts of the world. In developing countries, 70 to 90% of the population carries H. pylori. In developed countries, the prevalence of infection is lower. There appears to be no substantial reservoir of H. pylori aside from the human stomach. Transmission can occur by iatrogenic, fecal-oral, and oral-oral routes. H. pylori is able to colonize and persist in a unique biological niche within the gastric lumen. All fresh isolates of H. pylori express significant urease activity, which appears essential to the survival and pathogenesis of the bacterium. A variety of tests to diagnose H. pylori infection are now available. Histological examination of gastric tissue, culture, rapid urease testing, DNA probes, and PCR analysis, when used to test gastric tissue, all require endoscopy. In contrast, breath tests, serology, gastric juice PCR, and urinary excretion of [15N]ammonia are noninvasive tests that do not require endoscopy. In this review, we highlight advances in the detection of the presence of the organism and methods of differentiating among types of H. pylori, and we provide a background for appropriate chemotherapy of the infection.

Genetic relatedness of Burkholderia (Pseudomonas) cepacia isolates from five cystic fibrosis centers in Michigan.

Burkholderia cepacia isolates from patients with cystic fibrosis (CF) attending five CF centers were studied for relatedness by cellular fatty acid methyl esters (FAME) and by chromosomal DNA restriction analysis. Twenty-eight of 32 (87.5%) isolates tested were grouped in cluster group 1 based on their FAME profiles. DNA analysis revealed that 29 of 32 (90.6%) B. cepacia isolates from five CF centers had one closely related DNA pattern. To examine strain variation over a time period, FAME profiles and DNA patterns of isolates from serial cultures on seven patients from center D were studied. For four patients, all serial B. cepacia isolates belonged to a single FAME cluster group; for the remaining three patients, all serial isolates belonged to any two of the four cluster groups. On serial culture isolates, a single DNA pattern (pattern A) was found in 31 of 32 isolates demonstrating a close genetic relatedness. These data corroborate the observations that the majority of patients colonised with B. cepacia in a CF center harbor strains genetically closely related as determined by FAME profiles and DNA patterns.

Use of telepathology for routine surgical pathology review in a test bed in the Department of Veterans Affairs.

BACKGROUND: Routine surgical pathology review by telepathology could be an important service component of multi-institutional pathology laboratory systems. Such service networks would increase access for rural hospitals without on-site pathologists to a broader range of pathology services on a daily basis. METHODS: In this clinical trial, we analyzed the diagnostic accuracy, deferral rates, and viewing times of two generalist pathologists using a hybrid dynamic/store-and-forward (HDSF) telepathology (TP) system to render diagnoses in real time on 200 consecutive surgical cases. The objective was to assess the efficacy of TP in providing diagnostic surgical pathology services to a remote hospital without an onsite pathologist. Surgical pathology specimens underwent gross preparation by specially trained personnel. When appropriate, this was done under the video supervision of a telepathologist. For TP, glass slides were placed on the stage of a robotic microscope at the Iron Mountain (MI) Department of Veterans Affairs Medical Center (VAMC) (remote site); control of the motorized microscope was then transferred to a pathologist located 220 miles away at the Milwaukee VAMC (host site). For each case, the telepathologist had the option of rendering a diagnosis or deferring the case for later analysis by conventional light microscopy (LM). After the slides were read by TP and a surgical pathology report had been generated, the slides were transported to Milwaukee, where they were reexamined by the telepathologist using LM and then by the pathology group practice or, when there was no consensus, by an outside consultant to establish a "truth" diagnosis. RESULTS: Compared with the consensus ("truth") diagnosis, clinically important and overall concordance were 99.0% and 97.4%, respectively, by TP, and clinically important and overall concordance were 100.0% and 98.5%, respectively, by LM. The deferral rate was 2.5%. Examining glass slides by HDSF telepathology took an average of 4.43 minutes per slide and 12.09 minutes per case. CONCLUSION: The high diagnostic accuracy and low rate of case deferral support the proposal that an offsite pathologist using HDSF telepathology can substitute effectively for an onsite pathologist as a service provider.

Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies.

Helicobacter pylori is a spiral, gram-negative bacterium which causes chronic gastritis and plays a critical role in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori expresses significant urease activity which is an essential virulence factor. Since a significant fraction of urease activity is located on the surface of the bacterium, the urease molecule is a logical choice as an antigen for a vaccine; currently recombinant urease apoenzyme is being tested as a vaccine in phase II clinical trials. We have recently demonstrated that urease and HspB (a homolog of the GroEL heat shock protein) become associated with the surface of H. pylori in vitro in a novel manner: these cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria, reflecting the unique characteristics of the outer membrane. To determine if similar mechanisms are operative in vivo, we determined the ultrastructural locations of urease and HspB within bacteria present in human gastric biopsies. Our results demonstrate that both urease and HspB are located within the cytoplasm of all bacteria examined in human gastric biopsies. Interestingly, a significant proportion of the bacteria examined also possessed variable amounts of surface-associated urease and HspB antigen (from 5 to 50% of the total antigenic material), indicating that in vivo, H. pylori has surface characteristics which enable it to adsorb cytoplasmic proteins. This is consistent with our altruistic autolysis model in which H. pylori uses genetically programmed bacterial autolysis to release urease and other cytoplasmic proteins which are subsequently adsorbed onto the surface of neighboring viable bacteria. These observations have important implications regarding pathogenesis and development of vaccines for H. pylori.

Synthesis and activity of Helicobacter pylori urease and catalase at low pH.

BACKGROUND: Helicobacter pylori produces large amounts of urease presumably to be prepared for the rare event of a sudden acid exposure. The hypothesis that H pylori is acid sensitive and protein production is inhibited by low pH was examined. METHODS: H pylori or its soluble enzymes were incubated buffered or unbuffered at a pH ranging from 2-7 in the presence of 5 mM urea for 30 minutes. After exposure, urease and catalase activities of whole cells, supernatants, and soluble enzyme preparations were measured at pH 6.8. Newly synthesised enzyme was quantified by immunoprecipitation of [35S]-methionine labelled protein. RESULTS: Exposure to buffer below pH 4 resulted in loss of intracellular urease activity. In soluble enzyme preparations and supernatant, no urease activity was measurable after incubation at pH < 5. In contrast, catalase in whole cells, supernatant, and soluble enzyme preparations remained active after exposure to pH > or = 3. Exposure below pH 5 inhibited synthesis of total protein including nascent urease and catalase. At pH 6 or 7, urease represented 10% of total protein, catalase 1.5%. Exposure of H pylori to unbuffered HCl (pH > 2) resulted in an immediate neutralisation; urease and catalase activities and synthesis were unchanged. CONCLUSION: Low surrounding pH reduces activity of urease and synthesis of nascent urease, catalase, and presumably of most other proteins. This suggests that H pylori is not acidophilic although it tolerates short-term exposure to low pH.

Dynamic-robotic telepathology: Department of Veterans Affairs feasibility study.

In this retrospective study, we assess the accuracy, confidence levels, and viewing times of two generalist pathologists using both dynamic-robotic telepathology and conventional light microscopy (LM) to render diagnoses on a test set of 100 consecutive routine surgical pathology cases. The objective is to determine whether telepathology will allow a pathology group practice at a diagnostic hub to provide routine diagnostic services to a remote hospital without an on-site pathologist. For TP, glass slides were placed on the motorized stage of the robotic microscope of a telepathology system by a senior laboratory technologist in Iron Mountain, MI. Real-time control of the motorized microscope was then transferred to a pathologist in Milwaukee, WI, who viewed images of the glass slides on a video monitor. The telepathologists deferred rendering a diagnosis in 1.5% of cases. Clinically important concordance between the individual diagnoses rendered by telepathology and the "truth" diagnoses established by rereview of glass slides was 98.5%. In the telepathology mode, there were five incorrect diagnoses out of a total of 197 diagnoses. In four cases in which the telepathology diagnosis was incorrect, the pathologist's diagnosis by LM was identical to that rendered by telepathology. These represent errors of interpretation and cannot be ascribed to telepathology. The certainty of the pathologists with respect to their diagnoses was evaluated over time. Results for the first 50 cases served as baseline data. For the second 50 cases, confidence in rendering a diagnosis in the telepathology mode was essentially identical to that of making a diagnosis in the LM viewing mode. Viewing times in the telepathology mode also improved with more experience using the telepathology system. These results support the concept that an off-site pathologist using dynamic-robotic telepathology can substitute for an on-site pathologist as a service provider.