A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes.

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress.

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.

Abdominal pain in geriatric emergency patients: variables associated with adverse outcomes.

OBJECTIVE: To determine the diagnoses and outcomes of geriatric patients with abdominal pain, and to identify variables associated with adverse outcomes. METHODS: Geriatric emergency patients (aged 65 years and older) with a complaint of abdominal pain were participants in this longitudinal case series. Eligible patients were followed by telephone contact and chart review, to determine outcomes and final diagnoses. RESULTS: Of 380 eligible patients, follow-up information was available for 375 (97%), for the two months following the ED visit. Final diagnoses included infection (19.2%), mechanical-obstructive disorders (15.7%), ulcers/hypersecretory states (7.7%), urinary tract disease (7.7%), malignancy (7.2%), and others. Although 5.3% of the patients died (related to presenting condition), most (61.3%) patients ultimately recovered. Surgical intervention was required for 22.1% of the patients. Variables associated with adverse outcomes (death, and need for surgical intervention) included hypotension, abnormalities on abdominal radiography, leukocytosis, abnormal bowel sounds, and advanced age. Most physical examination findings were not helpful in identifying patients with adverse outcomes. This study demonstrated a higher incidence of malignancy (7.2%) and a lower incidence of disease necessitating surgical intervention (22.1%) than previously reported. CONCLUSIONS: The majority of geriatric emergency patients with abdominal pain have significant disease necessitating hospital admission. Morbidity and mortality among these patients are high, and specific variables are strongly associated with death and the need for surgical intervention. Absence of these variables does not preclude significant disease. Physical examination findings cannot reliably predict or exclude significant disease. These patients should be strongly considered for hospital admission, particularly when fever, hypotension, leukocytosis, or abnormal bowel sounds are present.

Skin necrosis and venous thrombosis from subcutaneous injection of charcoal lighter fluid (naptha).

A 33-year-old white man injected approximately 4 cc of charcoal lighter fluid (99.4% naptha/0.6% inert ingredients) subcutaneously into his left antecubital fossa. The injection resulted in the toxic necrosis of his volar forearm skin extending proximally to mid-humerus and distally to the metacarpophalangeal joints of the left hand dorsally over a 6-day period. The patient ultimately required extensive surgical debridement, secondary operative closure, and approximately 150 cm2 of split-thickness skin grafting. This case demonstrates the potential for widespread, delayed toxic necrosis of the skin resulting from subcutaneous injection of naptha. This patient's case appears to represent the most severe and widespread case of toxic necrosis of the skin resulting from the subcutaneous injection of hydrocarbons reported in the literature. This case also demonstrates extensive toxic thrombophlebitis not reported in prior cases involving subcutaneous injection of hydrocarbons.

A splice variant of trkB and brain-derived neurotrophic factor are co-expressed in retinal pigmented epithelial cells and promote differentiated characteristics.

There is evidence suggesting reciprocal trophic interactions between photoreceptors and the retinal pigmented epithelium (RPE), but the factors involved have not been identified. In this study, we investigated the hypothesis that one or more known neurotrophic factors act upon the RPE. Cultured human and freshly isolated bovine RPE cells demonstrated saturable specific binding for [125I]labeled BDNF, NT-4/5 and NT-3 with little specific binding for CNTF and none for NGF. Cross-competition experiments showed that BDNF is the preferred ligand and cross-linking of [125I]BDNF resulted in a doublet at 160 kd that was increased in RPE cells incubated in all-trans retinoic acid. There was basal phosphorylation of a 145 kd protein recognized by an anti-trk antibody that was increased in RPE cells pulsed with BDNF. RT-PCR with primers spanning the transmembrane domain demonstrated that RPE cells express trkB mRNA lacking a region homologous to exon 9 of chicken trkB, a splice variant that has been demonstrated to preferentially interact with BDNF. Northern blots demonstrated that cultured RPE cells also express mRNA for BDNF. BDNF did not stimulate proliferation or increase survival of RPE cells in serum-free medium, but promoted a differentiated morphology and increased the expression of cellular retinaldehyde binding protein, a marker of the differentiated state in RPE cells. An RPE cell line that spontaneously shows differentiated features showed a high level of BDNF mRNA. These data demonstrate that RPE cells express a short splice variant of trkB whose activation correlates with expression of differentiated characteristics and the cells themselves are capable of producing a ligand for the receptors. Signaling through trkB could play a role in differentiation of RPE cells during development and maintenance of the differentiated state in adult RPE.

Ethical issues of cardiopulmonary resuscitation: current practice among emergency physicians.

OBJECTIVE: To determine current practice and attitudes among emergency physicians (EPs) regarding the initiation and termination of CPR. METHODS: An anonymous survey was mailed to randomly selected EPs. Main outcome measures included respondents' answers to questions regarding outcome of resuscitation, and current practice regarding initiation, continuation, and termination of resuscitation for victims of cardiopulmonary arrest. RESULTS: The 1,252 respondents were from all 50 states, a variety of practice settings, and varying board certification. Most (78%) respondents honor legal advance directives regarding resuscitation. Few (7%) follow unofficial documents, or verbal reports of advance directives (6%). Many (62%) make decisions regarding resuscitation because of fear of litigation or criticism. A majority (55%) have recently attempted numerous resuscitations despite expectations that such efforts would be futile. Most respondents indicated that ideally, legal concerns should not influence physician practice regarding resuscitation (78%), but that in the current environment, legal concerns do influence practice (94%). CONCLUSIONS: Most EPs attempt to resuscitate patients in cardiopulmonary arrest, regardless of futility, except in cases where a legal advance directive is available. Many EPs' decisions regarding resuscitation are based on concerns of litigation and criticism, rather than their professional judgment of medical benefit or futility. Compliance with patients' wishes regarding resuscitation is low unless a legal advance directive is present. Possible solutions to these problems may include standardized guidelines for the initiation and termination of CPR, tort reform, and additional public education regarding resuscitation and advance directives.

Neurotrophic factors, cytokines and stress increase expression of basic fibroblast growth factor in retinal pigmented epithelial cells.

Basic fibroblast growth factor (bFGF) and FGF receptors have been localized to photoreceptors and retinal pigmented epithelium (RPE), but the function of bFGF in adult retina and RPE is unknown. Exogenous bFGF has a neuroprotective effect in retina and brain and its expression is increased in some neurons in response to cytokines or stress. In this study, we investigated the effect of light, other types of stress, neurotrophic factors, and cytokines on bFGF levels in cultured human RPE. Some agents that protect photoreceptors from the damaging effects of constant light, including brainderived neurotrophic factor (BDNF), ciliary neurotrophic factor, and interleukin-1 beta, increase bFGF mRNA levels in RPE cells. Intense light and exposure to oxidizing agents also increase bFGF mRNA levels in RPE cells and cycloheximide blocks the increase. An increase in bFGF protein levels was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that bFGF is modulated in RPE cells by stress and by agents that provide protection from stress and support the hypothesis that bFGF functions as a survival factor in the outer retina.

Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease.

Vascular endothelial growth factor (VEGF) is induced by hypoxia and it has been implicated in the development of iris and retinal neovascularization (NV) in ischemic retinopathies in which it has been suggested that Muller cells are responsible for increased VEGF production. VEGF, however, is also known to be a potent mediator of vascular permeability in other tissues and may perform this function in retina. Immunohistochemical staining for VEGF was performed on a variety of human and experimental ischemic and non-ischemic ocular disorders in which blood retinal barrier (BRB) breakdown is known to occur to determine if there is an upregulation of VEGF in these conditions. We found increased VEGF immunoreactivity in ganglion cells of rats with oxygen-induced ischemic retinopathy and in ganglion cells, the inner plexiform layer, and some cells in the inner nuclear layer of rats with experimental autoimmune uveoretinitis (EAU), in which there was no identifiable ischemia or NV. In rats with EAU, VEGF staining intensity increased from 8 to 11 days after immunization, coincident with BRB failure. These results were confirmed using two distinct anti-VEGF antibodies and by immunoblot and the immunohistochemical staining was eliminated by pre-incubating the antibodies with VEGF peptide. VEGF staining was also increased in the retina and iris of patients with ischemic retinopathies, such as diabetic retinopathy and retinal vascular occlusive disease, and in patients with disorders in which retinal ischemia does not play a major role, such as aphakic/ pseudophakic cystoid macular edema, retinoblastoma, ocular inflammatory disease or infection, and choroidal melanoma. VEGF was primarily localized within retinal neurons and retinal pigmented epithelial cells in these cases. In addition or in association with its role of inducing NV, VEGF may contribute to BRB breakdown in a variety of ocular disorders and blockage of VEGF signaling may help to reduce some types of macular edema.

Localisation of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes.

AIMS/BACKGROUND: Epiretinal membranes (ERMs) arise from a variety of causes or, in some cases, for unknown reasons. Once established, ERMs tend to progress, becoming more extensive and exerting increasing traction along the inner surface of the retina. One possible cause for their progression is the production of growth factors by cells within ERMs that may provide autocrine or paracrine stimulation. Platelet derived growth factor (PDGF) and its receptors have been localised to cells of ERMs and may play such a role. In this study, comparative data were sought for several other growth factors that have been implicated in ERM formation. METHODS: Immunohistochemical staining of ERMs was done for PDGF-A, PDGF-B, basic fibroblast growth factor (bFGF), three isoforms of transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) and its receptors, flt-1 and flk-1/KDR. Expression of flt-1 and flk-1/KDR was examined in cultured retinal pigmented epithelial (RPE) cells and retinal glia from postmortem eyes by immunohistochemistry and by reverse transcription coupled to polymerase chain reaction (RT-PCR). RESULTS: Staining was most intense and most frequently observed for VEGF and PDGF-A, both in vascular and avascular ERMs. The majority of cells stained for VEGF in nine of 11 (81.8%) diabetic ERMs and in 14 of 24 (58.3%) proliferative vitreoretinopathy ERMs. The receptors for VEGF, flt-1, and flk-1/KDR were also identified on cells in ERMs and on cultured RPE cells. By RT-PCR, mRNA for flt-1 was identified in RPE cells and retinal glia, and mRNA for flk-1/KDR was identified in RPE cells. CONCLUSIONS: These data show that VEGF and its receptors are localised to both vascular and avascular ERMs and suggest that VEGF, like PDGF-A, may be an autocrine and paracrine stimulator that may contribute to progression of vascular and avascular ERMs.

Hyalocytes synthesize and secrete inhibitors of retinal pigment epithelial cell proliferation in vitro.

BACKGROUND: Retinal pigment epithelial (RPE) cells that enter the vitreous in pathologic conditions, such as retinal detachment, may proliferate and contribute to the formation of epiretinal membranes. OBJECTIVE: To study whether hyalocytes, endogenous vitreous cells, play a role in modulating the proliferation of RPE cells. METHODS: Cell proliferation was measured by tritiated thymidine incorporation in density-arrested human RPE cells after incubation with media that had been conditioned by cultured bovine hyalocytes. Preliminary characterization of inhibitory activity in hyalocyte-conditioned medium was performed, including blocking experiments with a neutralizing antibody to transforming growth factor-beta 2 (TGF-beta) and proliferation assays that used MV-1-Lu mink lung epithelial cells. Northern blots were done to asses hyalocyte expression of TGF-beta messenger RNA. RESULTS: Hyalocyte-conditioned medium inhibited tritiated thymidine incorporation in RPE cells and MV-1-Lu mink lung epithelial cells in the presence or absence of serum or protease inhibitors. A portion of the inhibitory activity was neutralized by an antibody directed against TGF-beta. Northern blots of hyalocyte RNA demonstrated the presence of messenger RNA for TGF-beta 2. These data suggest that TGF-beta is responsible for a portion of the inhibitory activity secreted by hyalocytes. Additional inhibitory activity is attributable to one or more low-molecular-weight molecules distinct from TGF-beta. CONCLUSIONS: Hyalocyte-conditioned medium inhibits RPE cell proliferation in vitro through TGF-beta and at least one other molecule. Production of these factors by hyalocytes in vivo could provide a deterrent for epiretinal membrane formation that may be perturbed under pathologic conditions.

Fever in geriatric emergency patients: clinical features associated with serious illness.

STUDY OBJECTIVE: To determine the clinical significance of fever in geriatric emergency department patients. DESIGN: Case series with follow-up. SETTING: Urban, university-affiliated community hospital. PARTICIPANTS: Consecutive patients over the age of 65 years who presented to the ED during a 12-month period with an oral temperature of 100.0 degrees F (37.8 degrees C) or higher. RESULTS: We considered the following features indicators of serious illness: positive blood culture(s), related death within 1 month of ED visit, need for surgery or other invasive procedure, hospitalization for 4 or more days, IV antibiotics for 3 or more days, and repeat ED visit within 72 hours for related condition. Four hundred eighty-nine patients were eligible for study. Of the 470 patients with complete follow-up data, 357 (76.0%) had indicators of serious illness. Clinical features found to be independently associated with serious illness included oral temperature of 103 degrees F (39.4 degrees C) or more, respiration rate of 30 or more, leukocytosis of 11.0 x 10(9)/L or more, presence of an infiltrate, and pulse of 120 or more. At least one indicator of serious illness was present in 63 of 128 patients (49.6%) with none of these independently predictive clinical features. The most common final diagnoses were pneumonia (24.0%), urinary-tract infection (21.7%), and sepsis (12.8%). CONCLUSION: Fever among geriatric ED patients frequently marks the presence of serious illness. All such patients should be strongly considered for hospital admission, particularly when certain clinical features are present. The absence of abnormal findings does not reliably rule out the possibility of serious illness.

Plasma specific gravity for identifying hypovolaemia.

To define ranges of plasma specific gravity useful for identifying volume depletion in older adults, plasma specific gravity was measured in 170 young adults (mean age 28 years) and 100 retirees (mean age 81 years), and ranges of values likely to be associated with volume depletion were defined. Subsequently, measurements of plasma specific gravity were made in 68 older emergency room (ER) patients (mean age 74 years), a few of whom had obvious reasons for being hypovolaemic, e.g. dehydrating diarrhoea, and these results were compared to those for the control groups. Ranges for plasma specific gravity useful for identifying volume depletion were designated as possible hypovolaemia (1.0265-1.0279), probable hypovolaemia (1.0280-1.0294), and hypovolaemia (> or = 1.0295). Using these definitions, there were more older ER patients compared to both young and old control group subjects, respectively, with probable hypovolaemia (21% vs. 5% and 8%; p < 0.03) and hypovolaemia (16% vs. 0% and 0%; p < 0.03). This study establishes ranges for plasma specific gravity for young and old adults likely to be associated with hypovolaemia, and shows that based upon measurement of plasma specific gravity, older ER patients may often be hypovolemic even in the absence of obvious fluid-wasting illnesses. Future studies are needed to identify the risk factors for hypovolaemia in ER patients, and more vigorously substantiate the findings of this study.

Performance of seven rapid radiographic processing solutions.

This study evaluated the performance of seven rapid radiographic processing products. A conventional automatic processing solution was evaluated as a standard of comparison. The products were analyzed with respect to image quality and speed. The results indicate that rapid manual processing generally involves some compromise of image quality as well as slower speeds with an accompanying increase in patient radiation exposure. Accordingly, there seems to be little motivation for the use of rapid manual processing solutions unless there is a compelling rationale in terms of time or convenience.

Radiopacity of light-cured posterior composite resins.

Radiographic images of teeth and restorations were used to evaluate the radiopacity of 11 light-cured posterior composite resins. The radiopacity of these composite resins provided enough variation on radiographs so that clinicians distinguished the images of the restoration from adjacent tooth structure.

Human semen relaxin and its correlation with the parameters of semen analysis.

The relaxin content of 92 normal semen samples and 85 semen samples selected at random was correlated to the parameters measured on routine semen analysis in those samples. The concentration of immunoactive relaxin in a sample of semen plasma did not correlate with any of the parameters commonly used for semen analysis: count, percent motility, grade of forward progression, volume, and morphology.

The effect of relaxin and prostaglandin E2 on the motility of human spermatozoa.

Relaxin and prostaglandin E2 (PGE2) are present in human semen and have been shown to affect sperm motility. The authors further examined the effects of porcine relaxin and PGE2 on the motility of human spermatozoa. A dose-response study revealed that PGE2 at a concentration of 25 micrograms/ml is most effective in improving the motility of washed human sperm. Relaxin (100 ng/ml), PGE2 (25 micrograms/ml), or the two combined have no effect on the motility of spermatozoa in fresh, normal semen, suggesting that the constituents of fresh semen are optimal for motility. Relaxin and PGE2 individually improve the motility of washed spermatozoa. However, relaxin, but not PGE2, improves the motility of sperm in semen incubated at 37 degrees C for 5 hours (aged). In contrast to the individual substances, a combination of relaxin + PGE2 has no effect on the motility of washed spermatozoa or aged spermatozoa, suggesting that these two substances antagonize each other's actions on sperm motility. The presence of both relaxin and PGE2 in seminal plasma with normal motility spermatozoa suggests that other factors in seminal plasma regulate the effects of these substances on sperm motility.

Effect of relaxin on human spermatozoa.

To study its effect on the motility of human spermatozoa, relaxin was added at different concentrations to human semen samples of various qualities as well as to washed spermatozoa. Relaxin in physiologic concentrations (10-100 ng/mL) had no significant effect on sperm motility in normal semen samples. However, the addition of relaxin to semen samples with low sperm motility significantly increased the motility. Addition of relaxin similarly increased the motility of spermatozoa from normal semen samples that were either aged or washed; the treatment resulted in a decrease in motility. When sperm motility was optimal, as in normal samples, addition of relaxin did not increase motility. However, in some situations of decreased motility, addition of relaxin resulted in improvement of spermatozoan motility. Relaxin may have clinical value in the treatment of male infertility.