Nitrate synthesis in the germfree and conventional rat.

Metabolic balance studies show that germfree and conventional Sprague-Dawley rats synthesize nitrate. Equivalent results for germfree and conventional rats eliminate the microflora as obligatory components of nitrate production. Nitrate synthesis appears to be a mammalian process.

mTOR kinase inhibition reduces tissue factor expression and growth of pancreatic neuroendocrine tumors.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

In vivo dosimetry of 4-aminobiphenyl in rats via a cysteine adduct in hemoglobin.

The feasibility of monitoring doses of 4-aminobiphenyl (ABP) via adduction to hemoglobin was investigated. Rats dosed with ABP (from 0.5 micrograms/kg to 5 mg/kg) formed a stable covalent hemoglobin:ABP adduct. Approximately 5% of a single dose was bound as hemoglobin:ABP; chronic dosing led to an accumulation of the adduct to a level 30 times greater than that found after a single dose. Facile in vitro hydrolysis of the adduct regenerated ABP, allowing detection at the sub-ng level. Human hemoglobin was also readily adducted, using N-hydroxy-ABP in vitro. The predominant site of adduction appeared to be the cysteine residue in hemoglobin. The use of such adducts as dosimeters for arylamine exposures in humans is discussed.

BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells.

Inhibiting the bromodomain and extra-terminal (BET) domain family of epigenetic reader proteins has been shown to have potent anti-tumoral activity, which is commonly attributed to suppression of transcription. In this study, we show that two structurally distinct BET inhibitors (BETi) interfere with replication and cell cycle progression of murine Myc-induced lymphoma cells at sub-lethal concentrations when the transcriptome remains largely unaltered. This inhibition of replication coincides with a DNA-damage response and enhanced sensitivity to inhibitors of the upstream replication stress sensor ATR in vitro and in mouse models of B-cell lymphoma. Mechanistically, ATR and BETi combination therapy cause robust transcriptional changes of genes involved in cell death, senescence-associated secretory pathway, NFkB signaling and ER stress. Our data reveal that BETi can potentiate the cell stress and death caused by ATR inhibitors. This suggests that ATRi can be used in combination therapies of lymphomas without the use of genotoxic drugs.

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence.

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.

Effectiveness of ciprofloxacin-polystyrene sulfonate (PSS), ciprofloxacin and ofloxacin in a Staphylococcus keratitis model.

PURPOSE: Staphylococcus aureus causes severe corneal infections that often result in corneal scarring and blindness. Presently, therapy often involves the use of a fluoroquinolone antibiotic. This study, employing an experimental rabbit model of Staphylococcus keratitis, compared the effectiveness of two commonly prescribed formulations of fluoroquinolones to an experimental formulation, ciprofloxacin with polystyrene sulfonate (ciprofloxacin-PSS). The ciprofloxacin-PSS formulation uses an ion exchange resin to aid in the delivery of drug to the cornea. METHODS: Early (4-9 h postinfection, PI) and late (10-15 h PI) therapies were studied, employing 5 groups: ciprofloxacin-PSS, ciprofloxacin, ofloxacin, PSS vehicle. and untreated. Dosing regimens were: every 30 min, 60 min, or a single drop applied at 9 h PI. Eyes were observed by slit lamp examination (SLE) and bacterial colony forming units (CFU) per cornea were determined. RESULTS: Early phase therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min were equally effective (P > or = 0.2880), decreasing CFU per cornea by >5 log. Ciprofloxacin was significantly more active than ciprofloxacin-PSS or ofloxacin (P < or = 0.0410) when applied as a single drop. Late therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min resulted in >3 log decrease in CFU per cornea relative to controls (P < or = 0.0001). CONCLUSIONS: Topical treatment of experimental Staphylococcus keratitis with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin was effective. The effectiveness of ciprofloxacin-PSS suggests that improved drug delivery systems employing an ion exchange resin could be useful in an ocular fluoroquinolone formulation.

Epidemiology of microsporidiosis: sources and modes of transmission.

Microsporidia are single-celled, obligate intracellular parasites that were recently reclassified from protozoa to fungi. Microsporidia are considered a cause of emerging and opportunistic infections in humans, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission. Persistent or self-limiting diarrhea are the most common symptoms associated with microsporidiosis in immune-deficient or immune-competent individuals, respectively. Microsporidian spores appear to be relatively resistant under environmental conditions, and species of microsporidia infecting humans and animals have been identified in water sources, raising concern about water-borne transmission. Sensitive and specific immunomagnetic bead separation and PCR-based methods are being developed and applied for detecting microsporidia in infected hosts and water sources for generating more reliable prevalence data. The most effective drugs for treating microsporidiosis in humans currently include albendazole, which is effective against the Encephalitozoon species but not against Enterocytozoon bieneusi, and fumagillin, which has broader anti-microsporidia activity but is toxic in mammals, suggesting a need to identify better drugs. Strategies to capture and disinfect microsporidia in water are being developed and include filtration, coagulation, chlorination, gamma-irradiation, and ozonation.

Pharmacokinetics of topically applied ciprofloxacin in rabbit tears.

Fluoroquinolones provide an important single antibiotic therapy for bacterial keratitis caused by Staphylococcus aureus and numerous gram-negative bacteria, including Pseudomonas aeruginosa. The pharmacokinetics of three ocular fluoroquinolones, norfloxacin (CHIBOXIN, Merck, Sharp & Dohme), ciprofloxacin (CILOXAN, Alcon Laboratories), and ofloxacin (OCUFLOX, Allergan Pharmaceuticals), have been studied in the human eye and in both the normal rabbit eye and rabbit models of keratitis. However, the pharmacokinetics of ciprofloxacin have not been previously studied in the tear film of rabbits. This study was done to determine the pharmacokinetics of topical ciprofloxacin in the rabbit tear film. Two drops of CILOXAN were applied to the eyes of normal rabbits. Tear samples were collected at 5, 10 and 30 minutes, and 1, 2, 4 and 6 hours after topical drug application. Tear samples were analyzed for ciprofloxacin concentrations by HPLC. Ciprofloxacin concentrations reached a peak at 5 minutes, then declined in a manner similar to that reported for norfloxacin and ofloxacin. The ciprofloxacin concentrations in tears were substantially higher throughout the length of the study than the MIC90 for most ocular pathogens including Staphylococcus aureus and Pseudomonas aeruginosa.

Screening of compounds for antimicrosporidial activity in vitro.

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.

Anaphylactic shock and its implication for nurses.

Anaphylactic shock can strike a patient at any time or place. It is sudden allergic response to an environmental situation, insect venom or medically-prescribed drug. Although some members of the public may present in the Accident and Emergency department with life-threatening symptoms, the situation is often most serious when a drug has been given. The mechanism of anaphylactic shock is discussed in this article as is the first line treatment. The immune system and the character of immunoglobulins are highlighted, and the role of the nurse in an emergency situation is given consideration. Legal implications of any action taken are looked at from the standpoint of litigation.

Successful use of a bulk laxative to control the diarrhea of tube feeding.

The greatly increased nutritional demands of the severely burned patient frequently require the use of tube feeding for enternal hyperalimentation. At a time when general patient morale is low and motivation needs to be maximally maintained, there is nothing so dispiriting as the distress of a painful perianal region and uncontrollable liquid stools. Attempts to control the diarrhea which frequently accompanies tube feeding by changing the formula or the method of administration or a wide variety of constipating drugs have all met with very limited success. Based on the clinical observation of a noted gastroenterologist (Bockus), we have administered a mucilagenous hydrophilic colloid bulk laxative (Metamucil) to patients on tube feeding formulae. The dosage and frequency are adjusted to individual patient needs, but average 7 g per liter of liquid formula. The results have been dramatic; namely, the virtual elimination of the diarrhea problem in our burn patients on enteral hyperalimentation by gastric tube feeding. Colonic transit time increases. The stools become formed but soft, cohesive but not adhesive. Perianal irritation does not occur. Neither does soilage of wound, dressings, or bed. No rebound constipation or obstructive symptoms have been encountered. We attribute this response to the same water binding mechanism that allows these colloids to prevent chronic constipation. Our patients may be given as much as 5,000 to 6,000 calories of tube feeding per day. Our patients are not distressed by diarrhea. Our nursing staff is relieved of the burden that entails.

Pulmonary cestodiasis in a cynomolgus monkey (Macaca fascicularis).

Cestodiasis in primates has been noted historically to occur quite frequently, although tissue damage and clinical signs may or may not be apparent. Larval cestodes, such as hydatid cysts or cysticercus cysts, are known to cause extensive tissue damage, while other larval cestodes, such as tetrathyridia, cause minimal damage to the host. This case history concerns an apparently healthy cynomolgus monkey that was part of a surgical study. During the study, all parameters were normal. The monkey died 3 hours postsurgery. The apparent cause of death was respiratory arrest. At necropsy, it was discovered there were 1- to 3-mm-diameter cysts, which on transection extruded 2- to 5-mm-long larvae. The larvae could not be positively identified; however, they resembled tetrathyridia of Mesocestoides sp.

A meta-analysis of the relation between cumulative exposure to asbestos and relative risk of lung cancer.

OBJECTIVES: To obtain summary measures of the relation between cumulative exposure to asbestos and relative risk of lung cancer from published studies of exposed cohorts, and to explore the sources of heterogeneity in the dose-response coefficient with data available in these publications. METHODS: 15 cohorts in which the dose-response relation between cumulative exposure to asbestos and relative risk of lung cancer has been reported were identified. Linear dose-response models were applied, with intercepts either specific to the cohort or constrained by a random effects model; and with slopes specific to the cohort, constrained to be identical between cohorts (fixed effect), or constrained by a random effects model. Maximum likelihood techniques were used for the fitting procedures and to investigate sources of heterogeneity in the cohort specific dose-response relations. RESULTS: Estimates of the study specific dose-response coefficient (kappa 1.i) ranged from zero to 42 x 10(-3) ml/fibre-year (ml/f-y). Under the fixed effect model, a maximum likelihood estimate of the summary measure of the coefficient (k1) equal to 0.42 x 10(-3) (95% confidence interval (95% CI) 0.22 to 0.69 x 10(-3)) ml/f-y was obtained. Under the random effects model, implemented because there was substantial heterogeneity in the estimates of kappa 1.i and the zero dose intercepts (Ai), a maximum likelihood estimate of k1 equal to 2.6 x 10(-3) (95% CI 0.65 to 7.4 x 10(-3)) ml/f-y, and a maximum likelihood estimate of A equal to 1.36 (95% CI 1.05 to 1.76) were found. Industry category, dose measurements, tobacco habits, and standardisation procedures were identified as sources of heterogeneity. CONCLUSIONS: The appropriate summary measure of the relation between cumulative exposure to asbestos and relative risk of lung cancer depends on the context in which the measure will be applied and the prior beliefs of those applying the measure. In most situations, the summary measure of effect obtained under the random effects model is recommended. Under this model, potency, k1, is fourfold lower than that calculated by the United States Occupational Safety and Health Administration.

Fractionation of sporogonial stages of the microsporidian Encephalitozoon cuniculi by Percoll gradients.

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.

Lifestyle determinants of cancer among Danish mastic asphalt workers.

Three reports suggest that asphalt workers, especially young mastic asphalt workers, in Denmark experience an increase in incidence and mortality from cancer and in mortality from other conditions. The methodology described in these reports raises questions about their validity and the data presented are limited and difficult to interpret. The cancers and the causes of death that are increased are highly correlated with those seen among men in the general population who use alcohol in excess, who smoke, and who engage in other risk-taking behaviors. The effects of these lifestyle causes of disease were largely not controlled in the reported studies. These behaviors, which cluster in young men, rather than exposure to asphalt fumes, probably caused the disease patterns that were reported. Policy makers who use epidemiologic results for risk assessment and regulation should do so with care. Working men and women sometimes die at high rates and their occupational exposures may or may not be responsible. The distinction is crucial if occupational health is to be improved. If asphalt workers die young from excessive drinking and smoking, we are not protecting their health by controlling asphalt fume exposures.

Discrimination between viable and dead Encephalitozoon cuniculi (Microsporidian) spores by dual staining with sytox green and calcofluor white M2R.

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled) Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.