Biomedical subjects

D A McPhee

Publications and source records attributed to D A McPhee.

At least 19 recordsLinked to original sources

PubMed1992

A new electron microscope positive staining method for viruses in suspension.

A new procedure for the positive staining of viruses in suspension, the Tokuyasu staining procedure (TSP), was evaluated using a non-enveloped virus, rotavirus; an enveloped virus, rubella virus and two glutaraldehyde-treated enveloped viruses, Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) as models. The TSP involves an initial staining of the virus with uranyl acetate (UA) followed by thin embedding in a mixture of UA and polyvinyl alcohol (PVA). Using aqueous UA for the TSP, a combination of positively and negatively stained particles was seen for both rotavirus and rubella virus. With glutaraldehyde-fixed HTLV-I and HIV-1, stain penetration did not occur and only negative staining was observed. The substitution of methanolic UA for aqueous UA in the TSP resulted in only positive staining of rotavirus and rubella virus. The change in procedure also resulted in stain penetration of the glutaraldehyde-fixed HTLV-I and HIV-1 to give positively stained particles. Some novel morphological features of rotavirus and rubella virus structure were observed by the TSP.

Glutaral↗

PubMed1992

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.

Amino Acid Sequence↗

PubMed1992

Bacterial lipopolysaccharide mediates the loss of CD4 from the surface of purified peripheral blood monocytes.

In this study we have looked at the effect of lipopolysaccharide (LPS) on the surface antigen expression of cultured monocytes. Monocytes were purified from peripheral blood mononuclear cells (PBMC) and cultured in the presence or absence of LPS. The cultured cells were then stained with anti-MO3, anti-IL-2R and anti-CD4 MoAbs. We have shown that freshly isolated monocytes are IL-2R- and MO3-negative and express CD4 in low density. After overnight culture, without LPS, the expression of these surface markers remained relatively unchanged. However, in the presence of LPS (1 microgram/ml) CD4 expression was reduced to undetectable levels while the expression of IL-2R and MO3 was induced to maximal density. This effect of LPS on monocyte surface antigen expression was demonstrated with LPS preparations from Escherichia coli, Salmonella typhi and Vibrio cholerae. Surface antigen expression after 7 days culture in medium supplemented with non-heat-inactivated serum was essentially as seen after overnight culture, with the exception that LPS-induced IL-2R expression was transient. The ability to prepare monocytes that maintained surface CD4 expression after overnight culture was donor dependent.

Antigens, Surface↗

PubMed1991

Antibodies to gp41 and nef in otherwise HIV-negative homosexual man with Kaposi's sarcoma.

A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.

Genes, nef↗

PubMed1991

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Agglutination Tests↗

PubMed1991

N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.

Amino Acid Sequence↗

PubMed1990

Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies.

An enzyme immunoassay (EIA) utilizing a synthetic peptide analogue of HIV gp41 (amino acids 579-599, RILAVERYLKDQQLLGIWGCS) as antigen was compared with two commercial assays (Genetic Systems, Abbott ENVACORE) for the ability to detect antibodies in the early stages of infection. Two panels, consisting of 96 sera from 15 people and 140 sera from 44 people seroconverting to HIV, were examined. In the first group the synthetic peptide assay (gp41 EIA) detected antibodies before the Genetic Systems EIA in seven out of 15 people and concurrently in the remaining eight. With the second panel the Abbott ENVACORE assay detected antibodies before the gp41 EIA in two out of 44 people while the gp41 EIA detected antibodies first in six out of 44. In the remaining 36 people antibodies were detected simultaneously by the two tests. The gp41 EIA usually detected anti-HIV antibodies before or concurrently with the two commercial assays examined suggesting that the epitope cluster represented by this peptide is recognized early in infection.

AIDS Serodiagnosis↗

PubMed1989

Comparison of core antigen (p24) assay and reverse transcriptase activity for detection of human immunodeficiency virus type 1 replication.

This report compares two assay systems for monitoring human immunodeficiency virus (HIV) replication in peripheral blood leukocyte cultures. A commercial enzyme-linked immunoassay detected core antigen (p24) in 80% of cell cultures from HIV-seropositive individuals, whereas 67% of the cell cultures produced detectable levels of reverse transcriptase activity. There were clearly three patterns of reverse transcriptase activity produced, two of which may evade detection without regular sampling and maintaining cell cultures for more than 4 weeks. Once established, core antigen levels remained high so that cell cultures could be confidently monitored by an intermittent screening regimen.

Cells, Cultured↗

PubMed1989

Human monoclonal antibodies to HIV-1: cross-reactions with gag and env products.

Human monoclonal antibodies were raised by in vitro immunization of normal human spleen cells with denatured HIV-1 and subsequent fusion to an Epstein-Barr virus (EBV) transformed cell line. Three monoclonals reacted with both gag-encoded p24 core protein and env-encoded gp41 transmembrane glycoprotein. The cross-reaction was confirmed by reactivity with p24- and gp41-derived recombinant peptides. The peptides share two amino acid sequences which may be the basis for the cross-reaction. Attempted epitope mapping using synthetic peptides was unsuccessful due to non-specific reactivity with the short linear peptides.

Antibodies, Monoclonal↗

PubMed1988

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

Agglutination Tests↗

PubMed1988

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma.

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Maturation of immunological reactivity in the fetal lamb infected with Akabane virus.

The development of cell-mediated immunological reactivity was studied in fetal lambs infected with Akabane virus. Examination of hepatic cells from fetuses between 40 and 75 days' gestation that had been infected via the transplacental route revealed inconsistent responses to Akabane, together with a uniform failure to respond to non-specific mitogens which contrasted with the behaviour of control, uninfected lambs. Following direct inoculation of fetal lambs with virus between 50 and 120 days' gestation, specific proliferative responses were observed on the part of the spleen cells from some. Direct challenge of fetal lambs of 4 months' gestation evoked cellular responses in lymph draining from the site of virus inoculation similar to those produced by challenge of adult sheep. The proliferative response of lymph-borne cells was substantially better if live, rather than inactivated, virus had been used.

Animals↗

PubMed1988

Transmission of Akabane virus from the ewe to the early fetus (32 to 53 days).

The role of the placental junction in AKA virus infection in the ewe was examined during the time when the chorionic villi were first becoming firmly attached to the maternal caruncles. The studies were made over 21 days covering the period between 32 and 53 days of pregnancy. Viral tropism in the fetal membranes and tissues of the fetuses was identified by virus isolation and immuno-fluorescence studies. Areas of virus replication were noted from 24 h post-inoculation in the fetal membranes and persisted in these tissues throughout the experiment. Viral antigen was first detected in the fetus from day 5 post-inoculation by virus isolation and immuno-fluorescence. From this time on, viral activity increased in specific areas of the fetus, notably in the brain and, to a lesser extent, the skeletal muscles. Gross pathological changes occurred in the fetuses between day 14 and day 21 post-inoculation (46 to 53 days gestation). Despite the relatively high titres of AKA virus present in the placental tissues and the developmental changes occurring in the fetus due to the virus, the placental junction continued to carry out its physiological function of maintaining pregnancy.

Animals↗

PubMed1988

Antibody competition studies with gold-labelling immunoelectron microscopy.

Competitive studies, involving neutralizing monoclonal antibodies (NTmAbs) to outercoat proteins of Akabane virus were performed by immunoelectron microscopy. The experiments were designed to determine whether the NTmAbs were directed against the same or spatially different epitopes. Characteristics of NTmAbs in direct and indirect gold-labelling studies were determined. It was found that the protein A method gave cross-contamination of the immuno-gold complexes whereas direct conjugation of the NTmAbs to gold probes gave clean, specific and intense labelling. Analysis of dilution curves confirmed that saturation of antigenic sites did not occur and secondly determined the optimum working dilutions for the conjugated probes. The data generated in the preliminary studies enabled reliable results to be obtained from the double-labelling competitive experiment. We found that the 2 NTmAbs were directed to either the same epitope or to 2 separate but neighbouring epitopes where the binding of one NTmAb inhibited the binding of the second. The results demonstrate that if reliable data is to be obtained in double-labelling immunoelectron microscopical studies then experiments must be meticulously designed.

Antibodies, Monoclonal↗

PubMed1988

Quantitation of infectious human immunodeficiency virus using a modified plaque forming assay.

This report describes a modified plaque forming assay which has been used for quantitation of infectious human immunodeficiency virus (HIV) in cell culture supernatant fluid. The number of infectious units determined by the plaque assay correlates closely with the 50% infectious dose determined by a conventional assay in cell culture. In contrast, particle associated reverse transcriptase (RT) activity correlates poorly with the amount of infectious HIV present in culture supernatant fluids.

Cell Line↗

PubMed1988

Biochemical and serological comparisons of Australian bunyaviruses belonging to the Simbu serogroup.

Comparative analysis of the structural and possible non-structural proteins of seven Simbu serogroup bunyaviruses isolated in Australia revealed them all to be similar in size to those of Bunyamwera virus, the prototype of the Bunyavirus genus. The molecular weights of the structural proteins for these bunyaviruses (Akabane, Aino, Tinaroo, Douglas, Peaton, Facey's Paddock and Thimiri viruses) were 193K to 205K (L), 103K to 125K (G1), 33K to 37K (G2) and 25K to 26K (N). Analysis of the virion RNA of three viruses (Akabane, Douglas and Facey's Paddock) showed them all to be similar to Bunyamwera virus RNA, apparent Mr values being 2.6 X 10(6) (L), 1.4 X 10(6) to 1.9 X 10(6) (M) and 0.24 X 10(6) to 0.42 X 10(6) (S). Host cell protein synthesis was switched off late during infection, revealing four structural proteins L, G1, G2 and N. Comparative analysis of these protein profiles in infected Vero cells showed each virus, although similar, to be unique and easily identified; this method of comparison was efficient and rapid compared to the difficulty in obtaining adequate amounts of purified virus for analysis. Additionally, for all viruses except Douglas, two to four possible non-structural proteins were identified, with an Mr range from 12K to 30K. The viruses Akabane and Tinaroo, which have previously been shown to cross-react by plaque inhibition virus neutralization tests, were readily distinguished in migration of the G1 glycoprotein and by analysis of plaque reduction virus neutralization data using linear regression analysis of the dose-response curves. Using these same analyses, the differences between Aino and Douglas viruses, also related by plaque inhibition, were even greater. Application of the biochemical analysis of virus-specified proteins and some serological comparisons identified a mixed pool of different viruses in two unknown isolates grouped as Simbu serogroup viruses, and further identified a potential teratogenic strain in one of the two pools.

Animals↗

PubMed1987

Experimental infection of bulls and cows with bluetongue virus serotype 20.

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.

Animals↗

PubMed1987

Bluetongue virus serotype 20: experimental infection of pregnant heifers.

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.

Animals↗