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Biomedical subjects

M Bray

Publications and source records attributed to M Bray.

At least 19 recordsLinked to original sources
PubMed1995

Fundamental limitations of [18F]2-deoxy-2-fluoro-D-glucose for assessing myocardial glucose uptake.

BACKGROUND: The glucose tracer analog [18F]2-deoxy-2-fluoro-D-glucose (FDG) is widely used for assessing regional myocardial glucose metabolism in vivo. The reproducibility of this method has recently been questioned because of a discordant affinity of hexokinase for its substrates glucose and 2-deoxyglucose. We therefore compared rates of glucose utilization simultaneously with tissue time-activity curves of FDG uptake before and after changes in the physiological environment of the heart. METHODS AND RESULTS: Isolated working rat hearts were perfused for 60 minutes with recirculating Krebs buffer containing glucose (10 mmol/L), FDG (1 microCi/mL), [2-3H]glucose (0.05 microCi/mL), and [U-14C]2-deoxyglucose (2-DG; 0.025 microCi/mL). Myocardial glucose uptake was measured by tracer ([2-3H]glucose) and tracer analog methods (FDG and 2-DG) before and after the addition of either insulin (1 mU/mL), epinephrine (1 mumol/L), lactate (40 mmol/L), or D,L-beta-hydroxybutyrate (40 mmol/L) at 30 minutes of perfusion and after acute changes in cardiac workload. Under steady-state conditions, myocardial rates of glucose utilization as measured by tritiated water (3H2O) production from metabolism of [2-3H]glucose, FDG uptake, and 2-DG retention were linearly related. The addition of competing substrates decreased glucose utilization immediately. The addition of insulin increased the rate of glucose utilization as measured by the glucose tracer but not as measured by the tracer analogs. The ratio of 3H2O release/myocardial FDG uptake increased by 111% after the addition of insulin, by 428% after the addition of lactate, and by 232% after the addition of beta-hydroxybutyrate. Epinephrine increased rates of glucose utilization and contractile performance, whereas there was no increase in glucose uptake with a comparable increase in workload alone. There was no change in the relation between the glucose tracer and the tracer analog either with epinephrine or with acute changes in workload. CONCLUSIONS: The uptake and retention of FDG in heart muscle is linearly related to glucose utilization only under steady-state conditions. Addition of insulin or of competing substrates changes the relation between uptake of the glucose tracer and FDG. These observations preclude the determination of absolute rates of myocardial glucose uptake by the tracer analog method under non-steady-state conditions.

Animals↗
PubMed1995

Influence of the sample preservation mode to assess the chronic toxicity of an industrial effluent.

To improve the preservation of effluent samples, the contribution of refrigeration, freezing, and freeze-drying techniques to maintain the toxic characteristics of the samples was evaluated. To achieve this evaluation one acute test, three long-term toxicity tests, a biochemical test (P450 induction), and a mutagenicity test (Ames test) were used on the same industrial effluent. Refrigeration and freezing demonstrated a similar capacity of preservation. However, freezing should be recommended when the delay between sampling and the start of the bioassay exceeds 48 hr. Freeze-drying did not prove to be useful in that case but this could be due to methodological problems. Nevertheless, this technique allowed the detection of mutagenic compounds because of its aptitude to concentrate effluent samples. For the toxic evaluation of the samples the advantages of the use of a battery of biotests were confirmed and it was noted that the cladoceran (Ceriodaphnia dubia) reproduction test and the algal (Selenastrum capricornutum) growth test had a higher sensitivity within the three tests used.

Animals↗
PubMed1994

Recognition of envelope protein by dengue virus serotype-specific human CD4+ CD8- cytotoxic T-cell clones.

We analyzed dengue virus-specific CD4+ CD8- cytotoxic T lymphocytes (CTL) at the clonal level to further understand their role in dengue virus infections. Stimulation of peripheral blood mononuclear cells from two dengue virus type 4 (D4V)-immune donors with live D4V or noninfectious D4V antigen generated 17 HLA class II-restricted CD4+ CTL capable of specific lysis of dengue virus antigen-treated autologous lymphoblastoid cell lines. Thirteen clones were D4V specific, three clones were cross-reactive for D2V and D4V, and one clone was cross-reactive for D1V, D3V, and D4V. Antigen recognition by six D4V-specific clones and three D2V- and D4V-cross-reactive clones was restricted by HLA-DR7. Five D4V-specific CD4+ CTL clones lysed autologous lymphoblastoid cell lines infected with a dengue virus-vaccinia virus recombinant containing the E gene of D4V, whereas three serotype-cross-reactive CTL clones did not. These results indicate that E-specific clones are serotype specific and HLA-DR7 restricted in these two donors and suggest that a common epitope on E protein may be recognized. E protein-specific CD4+ CTL may be important mediators of virus clearance especially during reinfection with the same serotype as that in primary infection by providing help for virus-specific antibody production and lysis of virus-infected cells.

Antigens, Viral↗
PubMed1993

Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3. CTL from H-2b mice recognized a serotype-cross-reactive epitope that was localized to NS4a or NS4b. CTL from H-2d mice recognized at least three epitopes: a serotype-specific epitope on one of the structural proteins, a serotype-cross-reactive epitope on NS3, and a serotype-cross-reactive epitope on NS1 or NS2a. Our findings demonstrate the limited recognition of dengue virus proteins by CTL from three inbred mouse strains and the predominance of CTL epitopes on dengue virus nonstructural proteins, particularly NS3. Since human dengue virus-specific CTL show similar patterns of recognition, these findings suggest that nonstructural proteins should be considered in designing vaccines against dengue.

Animals↗
PubMed1992

Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.

Amino Acid Sequence↗
PubMed1991

Construction of intertypic chimeric dengue viruses by substitution of structural protein genes.

Dengue virus contains an 11-kilobase positive-strand RNA genome that codes for, in one open reading frame, three structural proteins (capsid, premembrane, and envelope), followed by seven nonstructural proteins. The structural protein genes of a full-length cDNA clone of type 4 dengue virus were replaced with the corresponding genes of dengue 1 or dengue 2 to create intertypic chimeric cDNA. The RNA transcripts made from these templates were infectious when transfected into permissive cells in culture. Progeny of chimeric cDNA produced apparently authentic dengue 1 or dengue 2 structural proteins, together with dengue 4 nonstructural proteins, and as a consequence exhibited type 1 or type 2 serological specificity. Both of the chimeras ultimately grew to the same titer as their type 1 or type 2 parent, but the type 2/type 4 chimera grew very slowly. This chimera also produced small plaques; in contrast, the type 1/type 4 chimera produced normal size plaques. The type 2/type 4 chimera retained the mouse neurovirulence of the dengue 2 virus, which was the source of its structural protein genes. Each of the mice inoculated intracerebrally with the chimera died, but survival time was prolonged. The retardation of replication of the type 2/type 4 chimeric virus suggests that this virus and possibly other intertypic dengue virus chimeras with similar properties should be examined for attenuation in primates and possible usefulness in a live dengue virus vaccine for humans.

Animals↗
PubMed1991

Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus.

Dengue virus is an enveloped positive-strand RNA virus with a genome approximately 11 kilobases in length. The four serotypes of dengue virus are currently the most important members of the flavivirus family in terms of geographical distribution and the incidence of infection in humans. In this communication we describe successful cloning of a stable full-length cDNA copy of dengue type 4 virus that can be used as the template for in vitro transcription of infectious RNA. Evidence is presented that dengue virus recovered from permissive cells transfected with the in vitro RNA transcripts retained a mutation that was engineered into full-length cDNA. The properties of the virus produced by cells transfected with infectious RNA transcripts of dengue cDNA resembled those of the virus from which the cDNA clone was derived. The dengue virus recombinant DNA system should prove helpful in gaining a better understanding of the molecular biology of dengue viruses and should facilitate the development of a safe and effective live vaccine for use in humans.

Base Sequence↗
PubMed1991

Dengue virus premembrane and membrane proteins elicit a protective immune response.

We have constructed recombinant vaccinia viruses that express the premembrane (pre-M), membrane (M), or the cleaved, residual portion of pre-M (non-M) proteins of dengue 4 virus, or the pre-M, non-M, or envelope (E) proteins of dengue 2 virus, to evaluate their ability to induce protective immunity in mice. Cells infected with these recombinants make proteins of expected size. Mice immunized with recombinants expressing dengue 4 pre-M or M were protected against subsequent dengue 4 encephalitis challenge, but non-M was not protective. However, a recombinant that expressed both pre-M and E as a polyprotein gave solid protection, while the simultaneous administration of the two recombinants expressing pre-M and E gave a significant level of protection. Pre-M and M function as antigens eliciting a protective immune response, and the combination of pre-M plus E is more protective than E alone.

Animals↗
PubMed1991

Carboxy-terminally truncated dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice.

Recombinant vaccinia viruses expressing C-terminally truncated E's that ranged in length from 9 to 99% of the N-terminal sequence were constructed. The overall antigenicity of the E products was analyzed by radioimmunoprecipitation, using dengue virus hyperimmune mouse ascitic fluid (HMAF) or an anti-E peptide serum. Truncated E that was 79% or less in length did not bind HMAF efficiently, whereas E constructs greater than 79% were able to bind HMAF with high efficiency. The first 392 amino acids of the dengue type 4 virus E sequence, including the Arg-392 following the 79% E C terminus, appeared to be critical for proper antigenic structure required for efficient binding by HMAF. Truncated E's ranging from 59 to 81% in length were secreted extracellularly, whereas smaller or larger E's were retained intracellularly. Secreted E's contained carbohydrate side chains that were resistant to endoglycosidase H digestion, suggesting that the transport of E occurs via a pathway from the rough endoplasmic reticulum through the Golgi complex. 79% E-RKG (which possessed the three additional amino acids immediately downstream of 79% E) was expressed at a high concentration on the surface of recombinant virus-infected cells presumably being inserted into the plasma membrane by a hydrophobic C-terminal membrane anchor. Evaluation in mice of the protective efficacy of the various vaccinia virus E recombinants indicated that only truncated E's that were recognized efficiently by HMAF induced a high level of resistance to dengue virus encephalitis. 79% E-RKG which is expressed at a high concentration on the surface of infected cells was highly immunogenic when tested for induction of an E antibody response. This suggests that cell surface expression of 79% E-RKG was responsible for its enhanced immunogenicity. Finally, passive immunization studies indicated that serum antibodies to E played a major role in the complete or nearly complete resistance to dengue virus challenge induced by certain vaccinia virus-truncated E recombinants.

Amino Acid Sequence↗
PubMed1990

Direct and indirect contractile responses of the human vas deferens and actions of noradrenaline and of calcium antagonists.

Contractile responses of the isolated human vas deferens, obtained from vasectomy operations, were measured. Large single electrical shocks gave a twitch response with short latency (0.36 s) which was insensitive to prazosin (5 microM) or TTX (0.2 microM) and was thus identified as due to direct muscle stimulation. A train of 100 low intensity shocks gave a response with a longer latency (1.9 s) which was substantially sensitive to both prazosin and TTX; we assume this response is dominated by an indirect nerve-induced contraction. Relaxations, presumably caused by activation of circular muscle, were recorded from regions of some preparations both by direct and indirect stimulation. Noradrenaline (10-20 microM) induced a tonic contracture, spontaneous contractions and a large potentiation of the response to direct stimulation--but not to indirect stimulation implying a strong presynaptic inhibition. Noradrenaline also speeded the relaxation from contractions. Verapamil (1-100 microM) and nifedipine had no effect on the direct responses but verapamil (10 microM) inhibited the indirect response. Calcium removal prevented most, and 5 mM-EDTA all, of the direct response. However, even with EDTA, noradrenaline was able to support spontaneous and stimulus-induced contractions. Thus contraction of the vas, though sustained by external calcium, does not appear to directly depend on it.

Calcium↗
PubMed1990

Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.

The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge.

Animals↗
PubMed1989

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes.

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.

Antibodies, Monoclonal↗
PubMed1989

Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.

We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.

Animals↗
PubMed1988

Fatal food-induced anaphylaxis.

Fatal food-induced anaphylaxis is rarely reported. In 16 months, we identified seven such cases involving five males and two females, aged 11 to 43 years. All victims were atopic with multiple prior anaphylactic episodes after ingestion of the incriminated food (peanut, four; pecan, one; crab, one; fish, one). In six cases the allergenic food was ingested away from home. Factors contributing to the severity of individual reactions included denial of symptoms, concomitant intake of alcohol, reliance on oral antihistamines alone to treat symptoms, and adrenal suppression by chronic glucocorticoid therapy for coexisting asthma. In no case was epinephrine administered immediately after onset of symptoms. Premortem or postmortem serum samples were available from six victims; in each case elevated levels of IgE antibodies to the incriminated food were demonstrated. Food-sensitive individuals must self-administer epinephrine promptly at the first sign of systemic reaction. Emergency care providers should be aware of cricothyrotomy as a life-saving procedure.

Adolescent↗
PubMed1988

Role of ionizing irradiation for 393 keloids.

Between 1928 and 1986, 393 keloid sites on 250 patients were presented. Three hundred seventy-five sites received superficial quality therapeutic irradiation. Etiologies were determined. The majority were excised prior to irradiation, and in these, histologic confirmation was obtained. Recurrence rate after irradiation was low, 9/375 (2.4%). When 7 of 9 recurrences are eliminated for post-irradiation ear lobe repiercing, true recurrence is only 2/375 (0.53%). Cosmetic result was considered excellent without recurrence in 92%; favorably improved with hypertrophic scar, nodule or altered pigment in 5.6%; and marginally improved with smaller, less symptomatic keloid in 2.4%. Significant complications were limited to persistent pigment disturbance in only 1 patient, and pre-irradiation hemorrhage in 1 patient (unrelated to radiotherapy). There was no wound dehiscence nor failure to heal irradiated incision site. Transient hyperpigmentation occurred in 91/375 (24%). Recurrence after irradiation was too rare to associate with etiology, specific technical factors, keloid configuration or velocity of initiating the post-operative radiotherapy. An unexpected finding was that 22/250 (9%) were Caucasian subjects. Our null (0%) carcinogenicity rate to date and the one-half of one percent true recurrence rate supports continued use of post-operative irradiation to prevent keloid formation in this group of documented troublesome keloid formers. Configuration subtypes are proposed, post-operative irradiation protocol is listed, and mechanism of action is explained in accordance with the dynamic pathophysiology of keloid.

Combined Modality Therapy↗