Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization.

Abstract

By using in situ hybridization methodology, we have directly examined primary lymph node and peripheral blood from patients with acquired immunodefIciency syndrome (AIDS) and AIDS-related complex for the presence of human T-lymphotropic virus type M (HTLV-l) viral RNA. Mononuclear cell preparations were hybridized with a 35s labeled HTLV-II-specific RNA probe and exposed to autoradiographic emulsion for 2 days. HTLV-III-infected cells expressing viral RNA were detected in '86% (6/7) oflymph node and 50% (7/14) of peripheral blood samples studied. However, in all patient samples eained, labeled cells were observed at very low frequency (<0.01% of total mononuclear cells). The HTLV-II-infected cells exhibited morphological characteristics consistent with that of lymphocytes and expressed viral RNA at relatively low abundance (20-300 copies per cell).These results demonstrate that HTLV-lI expression in lymph node and peripheral blood is very low in vivo. Furthermore, the lymph node hyperplasia observed in HTLV-Il-associated lymphadenopathy is not directly due to proliferation of HTLVrn-infected lymphocytes.The acquired immunodeficiency syndrome (AIDS) presents a severe unexplained immune deficiency that involves reduction in the number ofhelper T lymphocytes (OKT4) (1-3). The disease is usually accompanied by multiple opportunistic infections and/or malignancies, the latter predominantly of the Kaposi sarcoma type (4). AIDS-related complex (ARC) encompasses milder forms and sometimes prodromal states of the disease, and it is characterized by other clinical manifestations, most frequently unexplained chronic lymphadenopathy or leukopenia involving helper T lymphocytes (1-4). Recent serologic and viral isolation studies have shown that the development of AIDS or ARC is due to infection with the human retrovirus, human T-cell lymphotropic virus type III (HTLV-III) (5-8). HTLV-III, a cytopathic virus, is included in the HTLV family because of multiple biological and structural properties in common with HTLV types I and II (reviewed in ref. 9), including: (i) tropism for lymphocytes; (ii) particular tropism for OKT4 helper T lymphocytes; (iii) magnesium-dependent reverse transcriptase of high molecular weight; (iv) induction of giant multinucleated cells in culture; (v) impairment of T-cell functions; (vi) immunological crossreactivity of some virally encoded proteins; (vii) double-spliced 3' terminal mRNA; (viii) relatively small major core protein (p24/p25); (ix) unique juxtaposition of p24/p25 to the NH2-terminal gag protein-i.e., absence of the gag gene-encoded phosphoprotein in this position; and (x) trans-acting transcriptional activity resulting from a specific protein encoded by a viral gene.By using cloned HTLV-III probes (10, 11), analysis of fresh tissue from AIDS and ARC patients was carried out for detection of viral sequences. Southern blot experiments detected HTLV-III viral DNA at low levels in fresh lymphoid tissue from a number, albeit a minority, ofpatients with AIDS or ARC (12). At the s...

Experimental Infection of Pregnant Cattle with Bluetongue Virus Serotype 11 between Postbreeding Days 21 and 48

“…7 Reaction mixtures included 200 µCi of 35 S-UTP (> 1,000 Ci/mmol) d and were incubated for 60 min at 37 C. RNA probes had an specific activity of 10 7 cpm/µg. In situ hybridization was carried out as previously described 12 with slight modifications. Frozen sections of tissues were rehydrated in PBS/5 mM MgCl 2 for 10 min and then acetylated in 0.25% acetic anhydride/100 mM triethanolamine (pH 8.0) for 10 min.…”

Variations in CD4 Expression by Human Monocytes and Macrophages and Their Relationship to Infection with the Human Immunodeficiency Virus

“…They were then coated with liquid emulsion (Ilford L4) and incubated in the dark for 2 to 4 days at 4 °C. Finally they were developed in D-19 developer (Kodak), fixed in Ilford Hypam, counterstained with haematoxylin and eosin and examined by light microscopy (Harper et al, 1986).…”

Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers.

“…Plasmids were linearized at the EcoRl and HindIII restriction sites and transcribed into mRNA-complementary (antisense) and mRNAlike (sense) probes using IIS-UTP (New England Nuclear, Wilmington, DE) and a riboprobe kit (Promega Biotec, Madison, WI) according to the manufacturer's suggestions. Hybridization was carried out overnight at 45°C as described by Harper et al (24) . Slides were washed twice (10 min at room temperature) in 2 x SSC (lx = 0.15 M NaCl, 0.015 M NaCitrate, pH 7.0) and four times (15 min, 55°C) in 0.25X SSC,1 mM EDTA,1 mM DTT Nonhybridized probe was digested with 40 FAg/ml RNase A (Sigma Chemical Co.) in 10 mM tris, pH 8, 0.3 M NaCl for 30 min at 37°C.…”