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Biomedical subjects

D T Imagawa

Publications and source records attributed to D T Imagawa.

At least 19 recordsLinked to original sources
PubMed1992

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

Avian Leukosis Virus↗
PubMed1992

HIV-1 in postmortem brain tissue from patients with AIDS: a comparison of different detection techniques.

OBJECTIVE: The presence of HIV-1 in postmortem brain tissue from 31 patients with AIDS and 12 HIV-1-negative controls was investigated. DESIGN: Most laboratories have access to the methods used. We readily applied in situ hybridization and immunohistochemistry to archival formalin-fixed paraffin-embedded (FFPE) brain specimens. METHODS: The techniques used to detect HIV-1 were explant culture, in situ hybridization with 35S-labeled polymerase (pol) gene riboprobes and immunohistochemistry with monoclonal antibody to gp41. RESULTS: HIV-1 was isolated from explant cultures in 13 out of 20 (65%) patients, whereas HIV-1-infected cells were detected in FFPE brain tissue from nine out of 26 (35%) patients examined by in situ hybridization and in seven out of 26 (27%) patients examined by immunohistochemistry. CONCLUSIONS: Although the isolation technique was the most sensitive of the three techniques tested, infected cells may be identified with in situ hybridization in conjunction with immunohistochemistry.

AIDS-Related Opportunistic Infections↗
PubMed1991

HIV-1 propagates in human neuroblastoma cells.

A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS.

Animals↗
PubMed1990

The detection of HIV by in situ hybridization.

A simplified method of in situ hybridization is described for the detection of HIV targets. This standardized method can be applied to sections of formalin-fixed paraffin-embedded tissues, cell blocks, and smears of cultured cells using 3H- or 35S-labeled DNA and 35S-labeled RNA probes. In order to use elevated stringencies in the hybridization and wash steps, tissue sections and cells are covalently bonded to silanated glass slides without their subsequent loss from the slides. Routine hematoxylin and eosin or Romanovsky's stains enable the identification of the cells detected by in situ hybridization. HIV-infected neuroblastoma and lymphoid cell lines, lymph nodes, bone marrow, kidney, as well as brain tissue from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex are used to demonstrate the generality of the procedure. The standardized method described widens the ease and applicability of in situ hybridization in the investigation of pathologic tissues with the use of diverse specimens and probes.

DNA Probes↗
PubMed1989

Human immunodeficiency virus type 1 infection in homosexual men who remain seronegative for prolonged periods.

Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Bone marrow examination in patients with AIDS and AIDS-related complex (ARC). Morphologic and in situ hybridization studies.

Bone marrow examinations were performed on 20 patients with acquired immune deficiency syndrome (AIDS) and 39 with AIDS-related complex (ARC). Fever of unknown origin and thrombocytopenia were common in ARC, but anemia and leukopenia were most frequent in AIDS. Changes in stromal cells and perivascular cuffing of plasma cells were found significantly more often in patients with AIDS than in those with ARC. Malignancies were common in both groups. Human immunodeficiency virus (HIV) nucleic acids were detected with the use of a 3H-labeled cDNA probe with an in situ hybridization method in 11 bone marrow samples (three ARC and eight AIDS). Most commonly positive cells were mononucleated, resembling lymphocytes and histiocytes. Endothelial cells, interdigitating reticulum cells, nucleated red blood cells, and immature myeloid cells also had positive results in some instances. The number of HIV-positive cells was not related to the size of the bone biopsies or the clinical diagnoses. The authors postulate that changes in the peripheral blood and bone marrow of these patients may be related to latent persistent infection with HIV.

AIDS-Related Complex↗
PubMed1989

Cross-sectional study of reverse transcriptase-inhibiting antibody as a marker of acquired immune deficiency syndrome.

A cross-sectional study of 128 individuals infected with human immunodeficiency virus type 1 (HIV-1) was conducted to determine the correlation of reverse transcriptase-inhibiting (RTI) antibody to clinical disease. Thirty-two individuals were studied in each of four clinical groups: asymptomatic individuals, those with persistent generalized lymphadenopathy, those with acquired immune deficiency syndrome (AIDS)-related complex, and those with AIDS. Our study showed that 78% of asymptomatic individuals, 53% of those with persistent generalized lymphadenopathy, 50% of those with AIDS-related complex, and only 25% of those with AIDS have RTI antibody. Concurrent measurement of measles antibody level was used as an indicator of the immune status of these individuals. Measles antibody did not decline in persons with clinical disease, but asymptomatic individuals had lower antibody titers, possibly due to hypergammaglobulinemia associated with advanced HIV infection. These results indicate that more HIV-infected asymptomatic individuals than symptomatic individuals have RTI antibody. This suggests either that the RTI antibody level decreases with the progression of disease in HIV infection or that symptomatic individuals do not produce RTI antibody. The presence or absence of RTI antibody can thus be used as a marker of advanced disease.

AIDS-Related Complex↗
PubMed1989

Demonstration of human immunodeficiency virus in renal epithelium in HIV-associated nephropathy.

HIV-associated nephropathy (HIVAN) is a renal disease characterized clinically by heavy proteinuria and renal failure and morphologically by severe and rapidly evolving focal and segmental glomerulosclerosis, tubular necrosis, interstitial edema, and ultrastructural cellular inclusions. In an attempt to elucidate its pathogenesis, we evaluated the role of direct viral (HIV) infection of renal epithelium with the use of a cDNA probe for viral nucleic acid and an immunoperoxidase-labeled antibody to p24 core protein. In 10 of 11 kidneys with HIVAN, nucleic acid was localized to glomerular and tubular epithelium, while only 2 of 4 kidneys from HIV-infected patients with immune complex glomerulonephritis were similarly affected, but with considerably less cellular involvement. Kidneys from patients with acquired immune deficiency syndrome but without renal disease had only rare cellular positivity. In all instances, the cDNA probe was more sensitive than anti-p24 immunoperoxidase. These data suggest a role for direct HIV infection of renal epithelial cells in the initiation and/or progression of HIV-associated nephropathy.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Comparable sensitivities for detection of human immunodeficiency virus by sensitive reverse transcriptase and antigen capture enzyme-linked immunosorbent assays.

The sensitive overnight reverse transcriptase and the antigen capture enzyme-linked immunosorbent assays were tested for their ability to detect human immunodeficiency virus. The two assays were able to quantitate as few as 75 to 130 virus particles. Thus, both tests offer similar sensitivities for determining the presence of the virus.

Antibodies, Viral↗
PubMed1987

Isoelectric point restriction of cerebrospinal fluid and serum IgG antibodies to measles virus polypeptides in multiple sclerosis.

Thirty consecutive isoelectric point (pI)-discrete IgG fractions were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and used to immune precipitate measles virus (MV) polypeptides. Most basic fractions were enriched in activity against nucleocapsid protein (NP), and to a lesser extent against hemagglutinin (H) protein; intermediate fractions were enriched in activity against H and fusion (F) proteins; and more anodic pI fractions were almost exclusively enriched in activity against the large (L) protein of MV. In MS there are marked differences between CSF and autologous serum in regard to antibody activity to MV. In contrast, there were similar profiles of antibody response to MV proteins in SSPE CSF and serum.

Chemical Precipitation↗
PubMed1987

Autologous and allogeneic antibody responses to canine distemper virus isolates from dogs with chronic neurological diseases.

We describe the antibody responses to three strains of canine distemper virus (CDV) isolated from dogs with chronic neurological disease in the Los Angeles area using the naturally occurring sera and cerebrospinal fluids (CSFs) of these animals as probes for comparison. CDV/CDE-2 was derived from a dog with chronic distemper encephalitis, and CDV/ODE-8 and CDV/ODE-10 were derived from dogs with old dog encephalitis. Sera and CSFs were used in autologous (same dog) and allogeneic (different dog) combinations to immune precipitate the [35S]-methionine-labelled H, P, NP, F1, and M polypeptides of the virus-infected cell cultures. The polypeptides were separated by SDS-PAGE and detected by fluorography. There was decreased recognition by the CSF and sera of the polypeptides of the viral isolates in several autologous as well as allogeneic combinations. It is concluded that the immune responses to the CDV strains are not identical, and it is likely that viral mutations occurred after the animals were infected. Some mutations may have contributed to the pathogenesis of distemper encephalitis in these animals and some may have occurred during subsequent passage of the viruses in cell culture. This may explain the decreased recognition of the polypeptides of the viral isolates by the CSF and sera.

Animals↗
PubMed1987

Humoral immune response in dogs with old dog encephalitis and chromic distemper meningo-encephalitis.

The humoral immune response in sera and cerebrospinal fluids (CSFs) of dogs with various forms of canine distemper virus (CDV)-induced encephalitis was assessed by immunoprecipitation of radiolabelled nucleocapsid, phosphoprotein, membrane (M), haemagglutinin and fusion proteins. Sera from vaccinated dogs and hyperimmune sera contained antibodies to all the above antigens. In two cases of old dog encephalitis the sera and CSFs showed a restricted response to the M protein of CDV, whilst in three other cases of old dog encephalitis, two cases of chronic distemper (meningo-) encephalitis and experimentally induced encephalitis the humoral immune response appeared to be directed primarily to the nucleocapsid, phosphoprotein and the M protein but not the haemagglutinin or fusion proteins. Precipitation of the M protein by most of the sera was observed only when the antigen had been prepared by in vitro translation.

Animals↗
PubMed1987

Antibody that inhibits human immunodeficiency virus reverse transcriptase and association with inability to isolate virus.

Most individuals infected with human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome, produce an antibody against the viral reverse transcriptase (RT). Our studies show that 67% of HIV-seropositive individuals (33 of 49) produced an antibody that specifically inhibited viral RT enzyme activity. We were able to isolate HIV from only 18% of these individuals (6 of 33). On the other hand, virus was readily isolated from 63% of HIV-seropositive individuals (10 of 16) who did not demonstrate this antibody. Further examination of this RT-inhibiting antibody and its role during virus infection is needed, as it may prove to be of diagnostic, prognostic, or therapeutic value in this study and treatment of acquired immune deficiency syndrome.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Sensitive reverse transcriptase assay to detect and quantitate human immunodeficiency virus.

A sensitive biochemical assay of viral reverse transcriptase (RT) was developed that is useful for both the detection and quantitation of human immunodeficiency virus (HIV), the agent responsible for acquired immune deficiency syndrome in humans. This assay gave a 20- to 40-fold increase in enzyme activity over the current method used for RT detection of HIV. The test is based on a previous biochemical study showing the unusual stability of avian oncornavirus RNA-dependent DNA polymerases at 30 degrees C for at least 2 days. Our study shows that the HIV polymerase is stable at 30 to 37 degrees C for up to 3 days. By using this sensitive RT assay, as few as 250 HIV virions can be quantitated directly in tissue culture medium. This assay should prove useful in studies in which the detection of HIV or the quantitation of the number of virions is required.

Cell Line↗
PubMed1984

Mechanism of persistence with canine distemper virus: difference between a laboratory strain and an isolate from a dog with chronic neurological disease.

The mechanism of persistence in culture was different with canine distemper virus (CDV) isolated from a dog with chronic neurological disease (ODE; old dog encephalitis) compared to a laboratory strain (CDV/Ond). CDV/Ond persistence was achieved after seven undiluted passages while CDV/ODE-8, the recent isolate, showed immediate persistence in Vero cells. Both persistent infections resisted challenge with lytic CDV/Ond but not with unrelated vesicular stomatitis virus. The medium from CDV/Ond infection showed interference, whereas the medium from CDV/ODE-8 infection did not. Ultracentrifugation of the CDV/Ond supernatant effectively removed the defective interfering (DI) particles. Fluorescent microscopy showed the presence of CDV antigen in the cytoplasm of both types of persistently infected cells. By electron microscopy, 1% of the CDV/Ond cells and 23% of the CDV/ODE-8 cells showed distemper viral nucleocapsids. Persistence of CDV/Ond appears to be due to classical DI particles, whereas persistence of CDV/ODE-8 appears to be due to cell-associated particles. The persistence of CDV in dogs with ODE may be due to this cell-associated phenomenon.

Animals↗