Development of an information-intensive structure-activity relationship model and its application to human respiratory chemical sensitizers.

Structure-activity relationship (SAR) models are recognized as powerful tools to predict the toxicologic potential of new or untested chemicals and also provide insight into possible mechanisms of toxicity. Models have been based on physicochemical attributes and structural features of chemicals. We describe herein the development of a new SAR modeling algorithm called cat-SAR that is capable of analyzing and predicting chemical activity from divergent biological response data. The cat-SAR program develops chemical fragment-based SAR models from categorical biological response data (e.g. toxicologically active and inactive compounds). The database selected for model development was a published set of chemicals documented to cause respiratory hypersensitivity in humans. Two models were generated that differed only in that one model included explicate hydrogen containing fragments. The predictive abilities of the models were tested using leave-one-out cross-validation tests. One model had a sensitivity of 0.94 and specificity of 0.87 yielding an overall correct prediction of 91%. The second model had a sensitivity of 0.89, specificity of 0.95 and overall correct prediction of 92%. The demonstrated predictive capabilities of the cat-SAR approach, together with its modeling flexibility and design transparency, suggest the potential for its widespread applicability to toxicity prediction and for deriving mechanistic insight into toxicologic effects.

Proliferation of a subpopulation of human peripheral blood monocytes in the presence of colony stimulating factors may contribute to the inflammatory process in diseases such as rheumatoid arthritis.

Apart from acting on hemopoietic progenitor cells, colony stimulating factors (CSFs) have been shown to be involved in the activation, survival, proliferation and differentiation of more mature cells of the monocyte/macrophage lineage. There is evidence that a proportion of human peripheral blood monocytes can proliferate in response to CSF-1, (also known as M-CSF) and granulocyte-macrophage-CSF (GM-CSF). CSFs have been shown to be at elevated levels in the synovial fluid of RA patients and thus local proliferation of monocyte/macrophage within an inflamed lesion may contribute to the local tissue hyperplasia evident in inflammatory conditions. Flow cytometric analysis of surface antigen expression and cytokine production in response to lipopolysaccharide stimulation has been used to characterise the proliferating subpopulation of monocytes. Further characterization and subsequent isolation of this subpopulation of monocytes may provide new and important information necessary in understanding inflammatory diseases such as rheumatoid arthritis, where local proliferation at the site of inflammation may be a key factor contributing to the chronicity of the disease.

Characterization of a CSF-induced proliferating subpopulation of human peripheral blood monocytes by surface marker expression and cytokine production.

The phenotype of a subpopulation(s) of human monocytes which has been shown to proliferate in vitro in response to macrophage colony-stimulating factor (M-CSF or CSF-1) and granulocyte-macrophage CSF (GM-CSF) is as yet unknown. To identify this proliferating subpopulation(s) we demonstrated first that DNA synthesis was occurring under culture conditions suitable for flow cytometric evaluation. Flow cytometric analysis of surface antigen expression identified that after 5 days of culture the proliferating subpopulation of monocytes expressed CD14, CD13, CD33, CD11b, CD11c, CD87, HLA-DR, CD45RO, and did not express CD86, CD34, CD80, CD4, CD16, and CD56. In addition, these proliferating monocytes (representing approximately 5% of total monocytes) were shown to produce the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha in response to lipopolysaccharide stimulation. Further characterization and subsequent isolation of this subpopulation of monocytes may provide new and important information necessary to understand inflammatory diseases such as rheumatoid arthritis, where local proliferation at the site of inflammation may be a key factor contributing to the chronicity of the disease.

Direct comparison of the effects of CSF-1 (M-CSF) and GM-CSF on human monocyte DNA synthesis and CSF receptor expression.

There is evidence that a proportion of human monocytes can proliferate in vitro in response to colony-stimulating factor-1 (CSF-1, also known as M-CSF) and granulocyte-macrophage CSF (GM-CSF). To determine whether there are differences in DNA synthesis responses to these CSF, a large study using purified human peripheral blood monocytes from 45 donors was performed under optimized culture conditions. In contrast to the consistent response to CSF-1, approximately 20% of donors have monocytes that do not respond or have a minimal DNA synthesis response to GM-CSF stimulation. However, analysis demonstrated that no statistically significant differences exist in the levels of CSF-1 and GM-CSF-stimulated proliferation in monocytes. In addition, CSF-1 receptor (CSF-1R) blocking experiments indicated that a proportion of the GM-CSF-induced DNA synthesis is due to endogenous levels of CSF-1. As a further comparison of the actions of the two CSFs, CSF-1R and GM-CSFR levels were measured by flow cytometry, and it was shown that GM-CSFR levels decreased within 5 days of culture, independent of the conditions examined. In contrast, CSF-1R levels at day 5 approximated those measured in uncultured monocytes. Whether the proliferating subpopulation(s) express one or both CSF receptors at the beginning or at the end of culture is as yet unknown. The information obtained in this study will be useful for the design of strategies to enrich for the subpopulation in question based on CSF receptor expression.

Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.

Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A.

An examination of serial urinalyses in patients with North American crotalid envenomation.

STUDY OBJECTIVE: To examine the incidence of abnormal urinalyses after rattlesnake envenomations and its association with bite severity and antivenom administration. METHODS: A retrospective review of data collected in a prospective manner for an experimental crotalid antivenom trial. Subjects were individuals with minimal to moderate North American crotalid envenomations. Incidence and characterization of abnormal urinalysis after crotalid envenomation is presented. Additionally, the relationship of abnormal urinalysis to bite severity is examined. A preliminary test of antivenom protein urinalysis interference was also conducted. RESULTS: Forty-three percent of the urinalyses reported prior to antivenom treatment had abnormalities. Thirty-three of 41 subjects (80%) had an abnormal urinalysis, defined as the presence of cells, blood, glucose, or protein, at some time during the 2-week period following envenomation. All but 3 of these subjects had urinalyses which returned to normal by 2 weeks postenvenomation. Fifteen of 22 subjects (68%) with minimal envenomations had an abnormal urinalysis at some time following envenomation, while 18 of 19 subjects (95%) with moderate envenomations had abnormal urinalyses (p < 0.05). In addition, high concentrations of antivenom added to urine were found to produce a positive urine dipstick test for protein. CONCLUSION: In our study of patients with minimal to moderate North American crotalid envenomations, there was a high incidence of abnormal urinalyses. The urine abnormalities tended to be more common with increased bite severity and more frequent during the first few hours following envenomation. Antivenom appearance in the urine could be responsible for some of our findings.

The Smad5 gene is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on human hematopoiesis.

Signals from transforming growth factor-beta (TGF-beta), a bifunctional regulator of the proliferation of hematopoietic progenitor cells, have been recently shown to be transduced by five novel human genes related to a Drosophila gene termed MAD (mothers against the decapentaplegic gene). We showed by reverse transcriptase polymerase chain reaction that the RNA from one homologue gene, Smad5, was present in the immortalized myeloid leukemia cell lines, KG1 and HL60, in bone marrow mononuclear and polymorphonuclear cells, as well as in purified CD34+ bone marrow cells. Therefore, we studied the role of this gene in the regulation of human hematopoiesis by TGF-beta. TGF-beta1 and TGF-beta2 significantly inhibited myeloid, erythroid, megakaryocyte, and multilineage colony formation as assayed in semisolid culture systems. The levels of Smad5 mRNA in CD34+ cells were decreased by antisense but not sense oligonucleotides to Smad5. Preincubation of CD34+ marrow cells with two sense oligonucleotides to Smad5 did not reverse the inhibitory effects of TGF-beta on hematopoietic colony formation. However, preincubation with two antisense oligonucleotides to Smad5 reversed the inhibitory effects of TGF-beta. These data show that the Smad5 gene is involved in the signaling pathway by which TGF-beta inhibits primitive human hematopoietic progenitor cell proliferation and that Smad5 antisense oligonucleotides can interrupt this signal.

Outcome study of prehospital patients signed out against medical advice by field paramedics.

STUDY OBJECTIVE: To describe the incidence and demographic data of prehospital patients who contact paramedics by way of the 911 system, refuse transport against medical advice (AMA), then call 911 and are subsequently reevaluated by paramedics in the following 48 hours. METHODS: We conducted a retrospective observational review of records using the San Diego County Quality Assurance Network database for prehospital providers. All paramedic 911 responses that made base hospital contact over a 3-month period were reviewed to identify patients who signed out AMA. The main outcome measure was to identify patients who signed out AMA and then called 911 again within 48 hours. The demographics, complaints, treatments, and dispositions of these patients are described. RESULTS: Of 6,512 total 911 responses reviewed, 443 (7%) involved patients who signed out AMA. Of these patients, 156 cases (35.2%) were listed as trauma and 287 (64.8%) were medical, with cardiac chest pain, seizure, and respiratory distress/shortness of breath the most frequently noted medical subcategories. Fifty-one (11.5%) such patients received treatment; 34 received dextrose, 12 naloxone, 4 albuterol, and 1 a splint. Patient names were available in 5,515, of the total 6,512 responses and 431 of the 443 AMA cases, permitting computer searching of reevaluations by paramedics. Of the 431 AMA patients for whom a name was available, 10 (2%) called 911 again within 48 hours. All 10 callbacks were made for a related chief compliant, and all 10 of these patients were transported (4 admitted to hospital, 1 died en route, 1 transferred to another facility, 4 discharged from the ED). Of these 10 patients, 7 (70%) were older than 65 years, compared with 17% of all AMA patients older than 65 years. CONCLUSION: On the basis of our findings, patients over the age of 65 years have a propensity to recontact paramedics and should be aggressively encouraged to seek emergency medical treatment. Future prospective studies should be mounted to examine at patient outcome and to assess why patients sign out AMA after making contact with paramedics.

Association of rattlesnake bite location with severity of clinical manifestations.

STUDY OBJECTIVE: To examine an association between bite location in cases of North American crotalid envenomation and the severity of clinical manifestations. METHODS: We conducted a retrospective review of prospectively collected data for an experimental trial of crotalid antivenom. Our subjects were otherwise healthy individuals with minimal to moderate North American crotalid envenomation. We compared the severity of envenomation for patients with digit bites distal to the proximal interphalangeal joint and bites more proximal using a previously developed and validated snakebite severity score. RESULTS: Thirteen subjects were classified as having distal bites and 24 as having proximal bites. At baseline (before antivenom administration), the distal group had a mean severity score of 2.9 +/- 1.1, whereas the proximal group had a mean severity score of 5.0 +/- 2.2 (P = .0024). Patients in the proximal group tended to demonstrate a more rapid initial decline in severity score after receiving antivenom than did the distal group. CONCLUSION: In minimal to moderate North America crotalid envenomation, patients who sustained bites on distal aspects the digits tended to experience less severe clinical manifestations of envenomation. It is possible that an isolated bite to the distal aspect of a finger is an early marker of minimal envenomation.

The effect of trauma on the ocular penetration of intravenous ciprofloxacin.

PURPOSE: To study the intraocular pharmacokinetics of intravenously administered ciprofloxacin following eye trauma. METHODS: Twenty-three New Zealand albino rabbits and 12 Yorkshire pigs each received a surgically induced scleral injury to the right eye. Following repair, each rabbit received a single 30-mg intravenous infusion of ciprofloxacin. Each pig received either two 200-mg doses or two 400-mg doses of intravenous ciprofloxacin given 12 hours apart. Vitreous and serum samples were harvested at 0.5, 1, 4, 6, and 12 hours after antibiotic administration in rabbits and 1 hour after the second dose in pigs. Bioassays for ciprofloxacin were performed on each sample, and results were statistically compared by t test. The untraumatized left eye in each animal served as a control. RESULTS: The mean vitreous concentration of ciprofloxacin in traumatized rabbit eyes was 0.37 microgram/ml. This level was sustained above levels in control eyes (0.18 microgram/ml) for at least 4 hours following antibiotic administration. In control eyes, intravitreal levels peaked at 1 hour. Mean vitreous concentrations +/- SD in traumatized pig eyes were 0.091 +/- 0.017 microgram/ml in swine that had received 200-mg doses of ciprofloxacin vs 0.312 +/- 0.153 microgram/ml in swine that had received 400-mg doses (P = .02). Mean vitreous concentrations of ciprofloxacin in control eyes were not affected by increasing dosage. CONCLUSION: In both animal models, experimental surgical trauma increased intravitreal ciprofloxacin delivery. In addition, systemically administered ciprofloxacin achieved intravitreous levels exceeding minimum inhibitory concentrations for common ocular pathogens, suggesting a role for ciprofloxacin in the prophylaxis of posttraumatic endophthalmitis.

Intravenous cefazolin in penetrating eye injuries. A swine model.

PURPOSE: A swine model of penetrating ocular trauma was used to determine the delivery of systemically administered cefazolin to the vitreous cavity of traumatized and nontraumatized eyes. METHODS: Thirty-one pigs received a scleral laceration to the right eye under anesthesia and then were given intravenous cefazolin every 8 hours. Seven pigs received nine doses at 17 mg/kg. Seven animals received three doses of 36 mg/kg, and six others received nine doses of this regimen. Six pigs received three doses of 79 mg/kg and five others received three doses of 190 mg/kg. RESULTS: Vitreous levels of cefazolin averaged 15.6 micrograms/mL in traumatized eyes but were less than 1 microgram/mL in control eyes of animals receiving three doses at 190 mg/kg (P < or = 0.025). Mean serum concentration in these animals was 49.3 micrograms/mL. Vitreous levels were less than 1 microgram/mL in traumatized and control eyes in animals given lower doses of cefazolin (range, 17-79 mg/kg) despite multiple treatments over 2 and 3 days. CONCLUSIONS: These data demonstrate that systemically delivered cefazolin achieves levels ten times the minimum inhibitory concentration for Staphylococcus epidermidis in injured eyes. Therapeutic intraocular levels can be obtained through intravenous dosing, provided that therapeutic serum concentrations are achieved.

Spore attachment and extracellular mucilage of aquatic hyphomycetes.

Stages in conidiun attachment to surfaces of Lemmoniera aquatica and Mycocentrospora filiformis (freshwater Hyphomycetes) were studied at the light microscope and scanning and transmission electron microscope levels. Sigmoid conidia of M. filiformis attach by pre-existing conidial mucilage at the spore pole and at a point along the conidial body. Tetraradiate conidia of L. aquatica attach by the thigmotropic release of mucilage at the tips of the three "arms";. Germination in both species is followed by the production of germ tubes, germ hyphae and appressoria. The chemical composition of the mucilage involved in attachment was determined by enzymatic studies and lectin-gold cytochemical studies. The major component was found to be acidic poly-saccharide, comprising mainly ß-1, 3-glucan, N-acetyl-D-glucosamine and N-acetyl-neuraminic acid. Variation in mucilage composition exists between the two species, among different structures of the same species, and on different regions of the same structure. This indicates that mucilage producton in the two species is a dynamic process.The ability to secure rapid spore attachment, often in turbulent condition, would be a competitive advantage to these saprobic fungi in the colonization of substrata.

The management of sharps in the emergency department: is it safe?

In this study, we observed the management of sharps by health care workers including physicians, nurses, technicians, and students in the Emergency Department of the University of California-San Diego Medical Center. Twenty-eight percent of 418 observed sharp utilizations were managed in such a way that excess risk was conferred to the user, another person, or both. Twenty-seven percent conferred excess risk to the user and 12% to another person. Twenty percent of 322 recappable needles were recapped using a two-handed technique; 64% were disposed of uncapped. Four sharps (1%) were inadvertently thrown in the trash. Of the 418 observed sharp utilizations, none resulted in a puncture wound, although the four that were thrown in the trash represent a very high risk of injury to others. Physicians were observed handling the highest percentage of sharps in manners associated with excess risk while technicians and students managed sharps with the least risk. Among sharps used on patients who were IV drug abusers with unknown HIV status, 29% (n = 28) were handled with excess risk to the user, another person, or both. Of 24 sharps used on known HIV-infected patients, there were no practices observed that subjected either the user or another person to excess risk.

Interstitial cystitis and the urethral syndrome: a possible answer.

A study was made of 20 patients fulfilling the criteria customarily used for the diagnosis of interstitial cystitis. A possible infective aetiology was sought by culture of bladder tissue, catheter and midstream specimens of urine, and urethral swabs by methods capable of detecting fastidious bacteria as well as aerobic pathogens. All bladder biopsies showed the histological appearances usually associated with interstitial cystitis, and bacteria were isolated from the catheter specimens and/or bladder biopsies of 12 patients. Eight of these isolates were fastidious bacteria, Gardnerella vaginalis (6) and Lactobacillus sp. (2). Fastidious bacteria were isolated from the midstream specimen of urine (MSU) and/or urethral swab of 6 other patients. The correlation of the histological and bacteriological findings supports the hypothesis of an infective aetiology and suggests that the so-called urethral syndrome and interstitial cystitis may be the earlier and later stages of the same disease process. The importance of early diagnosis of infection in these patients is emphasised.

The thraustochytrids: a protist group with mixed affinities.

The thraustochytrids, a group of marine, monocentric protists are reconsidered phylogenetically drawing upon ultrastructural and biochemical characters. They appear to have affiliations with both heterokont groups and other phyla of marine organisms, but still remain an essentially independent entity.