Biomedical subjects
S Thompson
Publications and source records attributed to S Thompson.
The response of symptomatic neurosyphilis to high-dose intravenous penicillin G in patients with human immunodeficiency virus infection.
BACKGROUND: Infection with the human immunodeficiency virus (HIV) may affect both the natural course of syphilis and the response to treatment. We examined the response to treatment with high-dose penicillin G in HIV-infected patients with symptomatic neurosyphilis. METHODS: Neurosyphilis was defined by reactivity in serum treponemal tests for syphilis, neurologic manifestations consistent with neurosyphilis, and a positive Venereal Disease Research Laboratory (VDRL) test on cerebrospinal fluid. We identified 11 HIV-infected patients with symptomatic neurosyphilis; 5 had been treated previously for early syphilis with penicillin G benzathine. Patients were treated with 18 million to 24 million units of penicillin G per day administered intravenously for 10 days. Cerebrospinal fluid was examined approximately 6 and 24 weeks after treatment, when the polymerase chain reaction and rabbit inoculation were used to detect Treponema pallidum. RESULTS: In four of the seven patients studied 24 weeks after treatment, the serum titers on rapid plasma reagin (RPR) testing decreased by at least two doubling dilutions, and four patients had reductions in the cerebrospinal fluid titers on VDRL testing or reverted to nonreactive results. In two patients there was no normalization or improvement in serum titers on RPR testing or cerebrospinal fluid titers on VDRL testing, cell counts, or protein concentrations. One patient relapsed with meningovascular syphilis six months after therapy. T. pallidum was detected by the polymerase chain reaction in cerebrospinal fluid from 3 of 10 patients before treatment, but in none of the 10 post-treatment specimens. CONCLUSIONS: In patients with early syphilis who are also infected with HIV, therapy with penicillin G benzathine may fail, and neurosyphilis may develop. The regimen of high-dose penicillin recommended for neurosyphilis is not consistently effective in patients infected with HIV.
Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis.
Normal and retrovirally transfected keratinocyte suspensions expressing either the beta-galactosidase gene or the human growth hormone (hGH) gene were transplanted into chamber-enclosed skin full-thickness wounds of Yorkshire pigs. Immunostaining of sequential skin biopsies obtained for 4 weeks after transplantation showed survival of the transplanted keratinocytes as well as expression of beta-galactosidase. Transfected keratinocytes were first seen in the neodermal portions of the wounds, then in the regenerating basal epidermal layer, and finally in the terminally differentiating cells of the stratum spinosum. When keratinocytes transfected with the hGH gene were transplanted into similar wounds, hGH was detected for 10 days in wound fluid. In contrast, hGH was detected in vitro for 47 days. Wounds transplanted with either transfected or normal keratinocytes restored the epithelial barrier function significantly faster than nontransplanted controls (P < 0.05). The study confirms the successful transplantation of keratinocyte suspensions, their reconstitution of the epidermis, and their acceleration of repair. Further, this apparently normal incorporation of genetically engineered transplanted keratinocytes expressing either beta-galactosidase or hGH suggests the possibility of introducing other genes expressing therapeutic proteins into wounds to favorably affect healing. Wound fluid detection of the expressed peptide provided early demonstration of successful transfer of the hGH gene.
Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver.
The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but there is confusion in the literature about which P450 forms are detected in human and rat liver microsomes by these and homologous alkoxyresorufins. High performance liquid chromatographic analysis confirmed that O-dealkylation to resorufin was the sole or predominant route of metabolism for both short-chain and long-chain alkoxyresorufins and benzyloxyresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible variant form, CYP1A1*, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarbitone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pentoxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzyloxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-propoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-propoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CYP1A1* and CYP2B2 did not discriminate between them. The results show that ratios between different alkoxyresorufin O-dealkylation (AROD) activities can be more useful than absolute values of single activities for identifying P450 forms. Anti-P450 antibody and furafylline inhibition of rat liver microsomal AROD confirmed that ethoxyresorufin was a selective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresorufin was not a selective probe for CYP1A in liver microsomes from untreated or PB-induced rats, however, where it was metabolised mainly by CYP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloxyresorufin were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced microsomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discriminate CYP1A2 from CYP1A1, CYP1A1* or CYP2C6, furafylline inhibition indicated that methoxyresorufin was a selective measure of CYP1A2 in uninduced and 3MC-induced rat liver microsomes but not in ISF- or PB-induced microsomes. In human liver microsomes, antibody inhibition and furafylline inhibition showed that ethoxyresorufin and methoxyresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolism was due mainly to the CYP3A subfamily but also involved CYP1A2 and CYP2A6.(ABSTRACT TRUNCATED AT 400 WORDS)
Photocleavable nitrobenzyl-protein conjugates.
We have developed methods to allow the reversible binding of up to 15 nitrobenzyl residues per bovine serum albumin molecule and show 95% of these residues can be removed by exposure to UV light for 10 min. The general non-specific coating method is presented by means of a model system but is applicable to a wide range of proteins with important biological functions. Potentially, any protein could be coated with sufficient photo-removable groups to inhibit its biological function. Its activity may then be restored at will by exposure to UV light removing the coupled 2-nitrobenzyl groups.
The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties.
The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the cellular defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growth-suppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) and a resultant block to protein synthesis initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetratricopeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably transfected NIH 3T3 cells with a eukaryotic expression plasmid containing p58 cDNA under control of the cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, growing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2 alpha phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one mechanism by which the protein induces malignant transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis.
A comparative study of constitutive and induced alkoxyresorufin O-dealkylation and individual cytochrome P450 forms in cynomolgus monkey (Macaca fascicularis), human, mouse, rat and hamster liver microsomes.
The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). In monkeys there were no sex-differences in untreated, phenobarbitone (PB)- or beta-naphthoflavone (BNF)-treated animals in AROD activities, or in individual P450 proteins detected by immunoblotting. Basal MROD and EROD activities varied by less than 7-fold between the five species, but the comparative pattern of basal MROD, EROD, PROD and BROD activities (the "MEPB profile") was very species-specific, with monkeys being similar to rats but different from man, mouse and hamster. The induction of AROD activities by PB and BNF was also highly species-specific. Monkeys expressed constitutive proteins immunorelated to the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A sub-families (human CYP2A6 cross-reacted with the anti-rat CYP2B1 antibodies used, and so CYP2A and CYP2B forms could not be separately identified in the monkey). Single constitutive immunoblot bands were identified in monkey for CYP1A (54 kDa), CYP2A/CYP2B (51 kDa) and CYP3A (51 kDa), respectively, but two strong (51 and 52 kDa) plus two weak (49 and 49.5 kDa) bands were shown for CYP2C. Human liver expressed CYP1A2 (54 kDa), CYP2A6 (51 kDa), CYP3A4 (50.5 kDa) and three CYP2C9-immunorelated protein bands (48, 50 and 54 kDa). In monkeys BNF induced the 54 kDa CYP1A protein and CYP1A-dependent MROD, EROD and PROD activities (18-, 15- and 6-fold increases in activity, respectively), whereas PB strongly induced the 51 kDa CYP2A/CYP2B protein but did not induce PROD activity. PB also induced non-constitutive CYP2A/CYP2B protein bands at 49 and 52 kDa in some monkeys. BROD activity was induced less that four-fold by either PB or BNF in monkeys. In conclusion, cynomolgus monkeys expressed a range of constitutive CYP1A, CYP2A or CYP2B, CYP2C and CYP3A proteins similar to man, and a range of AROD monooxygenase reaction rates similar to both man and rat, but the basal MEPB profile of AROD activities in monkeys was more similar to rat than to man. MROD and EROD were good measures of CYP1A induction by polycyclic aromatic hydrocarbons in cynomolgus monkeys, but neither PROD nor BROD were indices of CYP2B induction by PB.
Care of pregnant women infected with HIV.
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A new percutaneous technique for establishing venous bypass access in orthotopic liver transplantation.
Partial veno-venous bypass (VVB) is commonly used in orthotopic liver transplantation (OLT). Venous access for blood return during VVB classically uses a surgical cutdown on the left axillary vein (LAV), which may prolong operating time and can be associated with significant complications. The authors have developed an alternative means of establishing venous access whereby the anesthesia team places 8.5F venous cannulae preoperatively in one or two vessels (internal jugular, antecubital, or subclavian) percutaneously using the Seldinger technique. These cannulae then serve to accept venous return from below the diaphragm via a centrifugal pump. The aim fo the present study was to compare the hemodynamic profiles obtained during the anhepatic phase of OLT in patients in whom either a conventional LAV catheter (group 1) or percutaneous catheters (group 2) were used for return flow from a centrifugal pump. There were no identifiable complications related to venous access in either group of patients. Total operating room time was 800 +/- 30 minutes in group 1 and 720 +/- 40 minutes in group 2 (P = 0.17). Hemodynamic parameters were determined from continuous strip chart recordings of arterial, right atrial, and inferior vena caval (IVCP) pressures. Cardiac output (CO) was measured by thermodilution whereas pump flow was determined by an electromagnetic probe. Renal perfusion pressure (RPP) was calculated as the difference between mean arterial pressure (MAP) and IVCP. Bypass pump flow was greater, but not significantly different between group 1 (3.0 +/- 0.2 L/min) and group 2 (2.4 +/- 0.2 L/min) (P = 0.09).(ABSTRACT TRUNCATED AT 250 WORDS)
Delayed hemolysis of white cell-reduced red cells.
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Aesthetic facial reconstruction: blending human perception and the facial subunit theory.
Facial reconstructive surgical results play a key role in how an individual accepts themselves and how society accepts the individual. Surgeons must strive to create the most aesthetic results, which includes reestablishing the expected contours, highlights, and landmarks. By understanding the subunits theory, plastic surgical nurses can provide patients with knowledgeable preoperative teaching, and can better anticipate the scope and necessary equipment for each reconstruction.
Integrated health and education input in the development of educational resources about asthma for schools.
The development of educational packages on health-related topics has become common in school curricula. This paper describes an integrated health and education input in the development of an educational package about asthma for Year 8 high school students. Ownership and educational relevance of the package (ensuring its appropriateness for inclusion within the Personal Development/Health/Physical Education curriculum) was achieved by collaboration between teachers with an understanding of the principles of curriculum design and health professionals with content knowledge about asthma. The model used for the production of the package about asthma could be extended to other health topics.
The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.
PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.
Detection of pooled secretions above endotracheal-tube cuffs: value of plain radiographs in sheep cadavers and patients.
OBJECTIVE: Mechanical ventilation is thought to increase the risk of nosocomial pneumonia by permitting leakage of bacteria-laden gastro-oropharyngeal secretions into the upper airways. The goal of this study was (a) to validate radiographic signs of pooled secretions above endotracheal-tube cuffs (supracuff liquid) in an animal model and (b) to determine whether suctionable pooled supracuff liquid can be identified on bedside radiographs of intubated patients. MATERIALS AND METHODS: Diagnostic criteria for supracuff liquid were initially validated by three radiologists interpreting 162 randomized radiographs made in an intubated sheep cadaver. The primary criteria included (a) replacement of the normal supracuff lucency with liquid opacity and (b) the formation of a sharp interface between the lucency of the upper edge of the cuff below and the liquid above. Graded infusions of 0, 3, 8, 13, and 23 ml of saline were made in triplicate into the space above the cuff, and radiographs were evaluated for the presence or absence of saline. The validated diagnostic criteria were used by two radiologists to estimate the frequency with which pooled liquid was seen on portable chest radiographs of 47 patients undergoing elective short-term postanesthetic mechanical ventilation. RESULTS: In the sheep-cadaver model, the diagnostic criteria for supracuff liquid allowed successful differentiation between no liquid, a small amount of liquid (3-8 ml), and a large amount of liquid (13-23 ml; c2, p < .0001). In a clinical study, radiographic signs of supracuff liquid were identified in 57% of 47 patients. In a small subset of patients (n = 18), the estimated liquid volume (mean +/- SEM) was calculated to be 7.8 +/- 1.1 ml (range = 2.1-18.4 ml). CONCLUSIONS: Radiography is a sensitive means of identifying small volumes of supracuff liquid above the inflated cuffs of endotracheal tubes. Potentially contaminating liquid pooled above the cuff of an endotracheal tube can be identified in about half of patients undergoing short-term mechanical ventilation. Our results suggest the suction of the supracuff space may be a reasonable prophylactic maneuver against nosocomial pneumonia. A much larger study is suggested to investigate the actual relation between pooled supracuff liquid and the development of nosocomial pneumonia.
Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro.
Self-cleaving RNAs (ribozymes) can be engineered to cleave target RNAs of choice in a sequence-specific manner (1). Consequently, they could be used to inhibit virus replication or to analyse host gene function in vivo. However, ribozymes that are catalytic in vitro are generally disappointing when analysed in cells unless expressed at high levels relative to their target RNAs (2, 3). Here we provide evidence that this can be overcome by optimizing ribozyme structure using cellular rather than cell-free assays. We show that ribozymes of relatively long flanking complementary regions (FCRs), while poor catalysts in vitro, can produce profound inhibition of HIV replication in cells. By examining a series of ribozymes in which the FCRs vary from 9 to 564 nucleotides, we establish that the optimum length for activity in the cell is > or = 33 nucleotides.
The glycosylation of haptoglobin in rheumatoid arthritis.
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Abnormally fucosylated haptoglobin as a marker for alcoholic liver disease but not excessive alcohol consumption or non-alcoholic liver disease.
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Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation.
The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). We report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22 t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH2 terminus of MLL, lacking the zinc-finger region, and that translocations occur in early hematopoietic cells, before commitment to distinct lineages.