Biomedical subjects
Publications and source records attributed to M R White.
In November 1995, a malignant mast cell tumor (mastocytoma) was diagnosed in an adult African hedgehog (Atelerix albiventris) from a zoological park (West Lafayette, Indiana, USA). The primary mast cell tumor presented as a firm subcutaneous mass along the ventrum of the neck. Metastasis to the right submandibular lymph node occurred.
Bacterial superinfections are a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. Depression of phagocyte functions resulting from attachment of the IAV hemagglutinin (HA) to cell surface sialo-glycoproteins is a likely contributory cause of these infections. We have proposed that the group of collagenous lectins (termed collectins) present in blood and pulmonary surfactant play a role in initial host defense against IAV. We used here several recombinant human surfactant protein D (RhSP-D) preparations to determine the mechanism through which opsonization of IAV with collectins protects neutrophils against the deactivating effects of IAV on cellular respiratory burst responses in vitro. RhSP-D was markedly more potent than antibodies that inhibited viral hemagglutination activity (anti-HA antibodies) at protecting neutrophils in this assay. Unlike the anti-HA antibodies, RhSP-D was protective at concentrations that minimally inhibited viral hemagglutination activity. Two related features of SP-D--the degree of multimerization and the ability to cause aggregation of IAV particles--were critical determinants of the ability of SP-D to protect neutrophils against deactivation. Similarly SP-D-induced viral aggregate formation resulted in enhanced IAV binding to neutrophils and potentiated the ability of the virus itself to trigger neutrophil respiratory burst responses. In contrast to the case of IAV-antibody complexes, SP-D-IAV complexes attached to and activated neutrophils through a neuraminidase-sensitive mechanism (ie, similar to unopsonized IAV). These results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.
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One-month-old Chinook salmon fry from a cold-water hatchery were presented live for euthanasia and necropsy. Gross lesions were emaciation in 90% of the fry and ascites and increased cutaneous pigmentation in the remaining 10%. A cause for the emaciation was not determined. Histologically, the fry with ascites and increased pigmentation had visceral mycosis with aerocystitis, myositis, peritonitis, and dermatitis. Sporobolomyces salmonicolor, a rare human pathogen, was isolated and identified in tissue sections from affected fry.
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OBJECTIVE: To determine whether administration of amphotericin B in a fat emulsion solution would reduce the nephrotoxicity of amphotericin B, compared with that associated with administration of amphotericin B in 5% dextrose solution. DESIGN: Prospective controlled study. ANIMALS: 2 groups of 5 adult male Beagles. PROCEDURE: Dogs received amphotericin B (1 mg/kg of body weight/d) prepared in 5% dextrose solution or in 20% fat emulsion daily for 6 doses. Serum biochemical analysis, CBC, urinalysis, and endogenous creatinine clearance was performed on days 0 and 8, 2 days after the last dose of amphotericin B. On day 8, dogs were euthanatized and gross necropsies were performed. Unbiased semiquantitative scoring of the kidneys for the degree of injury was performed by use of light microscopy. RESULTS: There were no significant differences in serum creatinine, urea nitrogen, or potassium concentrations, urine specific gravity, endogenous creatinine clearance, or degree of tubulo-interstitial injury between the 2 groups. CONCLUSION: In this model, the degree of nephrotoxicity of amphotericin B was not significantly different for dogs receiving the drug in a fat emulsion versus its administration in 5% dextrose.
BACKGROUND: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis of luciferase expression in single cells has previously been hampered by the insensitivity of existing methodologies and the lack of a way of monitoring quantitatively, and independently, more than one promoter within the same cell. We have developed a technique for examining the dynamics of insulin-stimulated AP-1-dependent transcription in single living cells, and have explored the signalling pathway involved. RESULTS: Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the comicroinjection of luciferase- and aequorin-based reporter plasmids directly into the cell nucleus, and the subsequent analysis of luminescence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene expression to be monitored in real time. We found that insulin promoted a substantial increase in the expression of a luciferase gene under the control of the AP-1-binding site from the collagenase gene promoter. Aequorin expression, under the control of a cytomegalovirus promoter, was unaffected by insulin. The effect of insulin on luciferase expression was specifically blocked by overexpression of either the mitogen-activated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS217/221A. CONCLUSIONS: Microinjection coupled with luciferase imaging allows hormone-regulated gene expression from relatively weak promoters to be monitored in single living cells. We have used this method to demonstrate that MAP kinase plays a central role in the ability of insulin to stimulate AP-1-dependent gene transcription.
Bacterial superinfections are the most common cause of mortality during influenza epidemics. Depression of phagocyte functions by influenza A viruses (IAVs) is a likely contributory cause of such infections. We used an in vitro model of viral depression of neutrophil respiratory burst responses to FMLP and PMA to examine the mechanism of IAV-induced phagocyte deactivation. Respiratory burst responses or intracellular calcium mobilization were triggered by the virus itself, but these were not causally related to deactivation. By treating neutrophils with neuraminidase, and by use of purified IAV hemagglutinin (HA) preparations, cross-linking of sialic acid-bearing neutrophil surface components by the IAV HA was shown to be responsible for deactivation. IAV competed for binding to neutrophils with Abs directed against CD43, sialyl-Le(x), CD45, and gangliosides. Deactivation could be reproduced by treating neutrophils with anti-CD43 or -sialyl-Le(x) Abs in the absence of IAV. However, treatment of neutrophils with elastase markedly reduced CD43 expression, without affecting overall IAV binding or the ability of IAV to cause deactivation. Hence, although IAV binding to CD43 can account for deactivation, other IAV-binding proteins exist (e.g., those bearing sialyl-Le(x)) that can independently mediate functional depression.
An important question for exobiology is, did life evolve on Mars? To answer this question, experiments must be conducted on the martian surface. Given current mission constraints on mass, power, and volume, these experiments can only be performed using proposed analytical techniques such as: electron microscopy, X-ray fluorescence, X-ray diffraction, alpha-proton backscatter, gamma-ray spectrometry, differential thermal analysis, differential scanning calorimetry, pyrolysis gas chromatography, mass spectrometry, and specific element detectors. Using prepared test samples consisting of 1% organic matter (bovine serum albumin) in palagonite and a mixture of palagonite, clays, iron oxides, and evaporites, it was determined that a combination of X-ray diffraction and differential thermal analysis coupled with gas chromatography provides the best insight into the chemistry, mineralogy, and geological history of the samples.
In order to accurately diagnose bacterial kidney disease caused by Renibacterium salmoninarum in steelhead trout, kidney tissue from experimentally infected fish was evaluated using a commercially available enzyme-linked immunosorbent assay (ELISA) test kit, fluorescent antibody (FA) testing, bacteriologic culture, and histopathology. Seventy-five steelhead trout were randomly assigned to 1 of 4 groups and intraperitoneally inoculated with 0.15 ml saline (n = 20), 1 x 10(10) organisms/ml (n = 18), 1 x 10(8) organisms/ml (n = 18), or 1 x 10(6) organisms/ml (n = 19) of R. salmoninarum. ELISA, FA and bacteriologic culture were positive for R. salmoninarum from the kidney tissue of the 2 groups infected with the highest doses. Although the ELISA and FA tests were accurate when compared to the bacteriologic culture from the 2 groups infected with higher doses of the organism, they were less sensitive at the lowest level of inoculum. Histopathology was not specific for this disease; however, all infected fish had a marked proliferative histiocytic interstitial nephritis, characterized by marked expansion of the renal hematopoietic tissue by histiocytes without tissue necrosis. Other microscopic findings included splenitis and myositis (at the injection site) of some fish.
The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.
The translation of a capped, polyadenylated RNA after injection into the nucleus of Xenopus oocytes occurs only if the RNA contains an intron. A single point mutation in the splice donor site prevents translation. Intron-less RNA is exported efficiently to the cytoplasm and is held, undegraded, in a translationally inert state for several days. Translation can be activated by treating the oocytes with progesterone or by injecting antibodies that bind the FRGY2 class of messenger RNA binding proteins, p56 and p60, but these antibodies are only effective if delivered to the nucleus. Inhibitors of casein kinase II also activate translation whereas phosphatase inhibitors block progesterone-mediated activation of translation. These data suggest the presence of an RNA handling pathway in the nucleus of Xenopus oocytes which is regulated by casein kinase type II phosphorylation and which directs transcripts to be sequestered by p56/p60 or by closely related proteins. This pathway can be bypassed if the RNA contains an intron and it can be reversed by progesterone treatment. These data may have implications for understanding translational control during early development.
Signalment, tumor sites, clinicopathologic, radiographic, and ultrasonographic features, as well as treatment protocols and survival information, were evaluated for 10 dogs with a histologic diagnosis of giant cell variant of malignant fibrous histiocytoma. Common clinical findings included subcutaneous masses, weight loss, anorexia, and lethargy. Laboratory abnormalities included anemia, hypoalbuminemia, and high concentrations of serum hepatic enzymes. Radiography and ultrasonography were useful in staging the extent of metastasis. Seven dogs had tumor metastasis at the time of diagnosis. Two other dogs developed evidence of metastasis during the course of treatment. The most common sites of tumor involvement were subcutaneous tissues, lymph nodes, liver, and lungs. Treatment protocols included surgical resection, intraoperative radiotherapy, and chemotherapy. Median survival time of all dogs was 61 days. Median survival time of the 6 treated dogs was 161 days. Findings on necropsy revealed metastasis with multiple organ involvement. The giant cell variant of malignant fibrous histiocytoma was determined to be a highly metastatic neoplasm in dogs, which may be responsive to surgical excision, chemotherapy, or radiotherapy.
We have studied in detail the in vitro interactions of influenza A viruses (IAVs) with human neutrophils to clarify why these cells become dysfunctional during IAV infection. Unosponized IAV elicited a respiratory burst response in neutrophils which, like that triggered by formylmethionyl-leucyl-phenylalanine (fMLP), involved mediation of signal-transducing GTP-binding proteins and tyrosine kinase activation. The IAV-induced response differed from that provoked by fMLP in that H2O2 was produced without concomitant O2- release. IAV also did not cause extracellular release of granule enzymes in cytochalasin B-treated neutrophils. Using chemiluminescence assays, the respiratory burst response to IAV was found to occur at an intracellular location. These findings may, in part, explain the anomalous nature of the respiratory burst response elicited by IAV and suggest strategies for determining the mechanism of IAV-induced neutrophil deactivation.
Despite increasing therapeutic use of interferon (IFN)-alpha, its effects on human neutrophil function are not well characterized. In vitro preincubation of neutrophils with recombinant IFN-alpha and -gamma, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced neutrophil respiratory burst responses to stimulation with influenza A virus (IAV) and FMLP. The enhancing effects of IFNs were more subtle and required more prolonged incubation than those of TNF and GM-CSF. TNF and GM-CSF enhanced neutrophil binding of IAV and neutrophil intracellular calcium and membrane depolarization responses to IAV or FMLP stimulation, while IFNs did not. Inhibitors of neutrophil tyrosine kinase activation and protein synthesis blocked IFN-alpha-induced enhancement of respiratory burst responses. In addition to its other well-characterized effects, IFN-alpha may protect against viral infection indirectly by promoting neutrophil respiratory burst responses.
Two experiments were conducted to estimate the dietary choline requirement and to determine the effects of dietary choline on liver lipid deposition in juvenile hybrid striped bass (Monrone saxatilis x M. chrysops). Experimental diets contained 0.73 g total sulfur amino acids/100 g diet (0.47 g methionine + 0.26 g cyst(e)ine/100 g diet), thus meeting, but not exceeding, the requirement. Graded levels of choline bitartrate in Experiment 1 and choline chloride in Experiment 2 were added to the basal diet, resulting in eight dietary treatments in each experiment. Dietary treatments were 0, 250, 500, 1000, 2000, 4000, 6000 and 8000 mg choline/kg dry diet. Diets were fed for 12 and 10 wk in Experiments 1 and 2, respectively. Dietary choline concentrations significantly affected weight gain, feed efficiency, survival and total liver lipid concentrations in each experiment. Weight gain and feed efficiency were greatest in fish fed 500 mg choline/kg dry diet as choline bitartrate. Total liver lipid concentrations were variable but tended to be lowest in fish fed diets containing at least 2000 mg choline/kg diet. Survival was significantly lower in the group of fish fed 8000 mg choline/kg diet supplied by choline bitartrate. Weight gain and feed efficiency were greatest and total liver lipid concentration was lowest in groups of fish fed at least 500 mg choline/kg diet as choline chloride; survival was unaffected by dietary treatment. Therefore, choline chloride seems to be a better source of dietary choline than choline bitartrate and 500 mg choline/kg diet is adequate for maximum weight gain and prevention of increased liver lipid concentration in juvenile hybrid striped bass.
Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community.
We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.