Birgitta Åsjö

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations

A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.

REPLICATIVE CAPACITY OF HUMAN IMMUNODEFICIENCY VIRUS FROM PATIENTS WITH VARYING SEVERITY OF HIV INFECTION

T-lymphotropic viruses were isolated from 31 patients with different clinical manifestations of human immunodeficiency virus (HIV) infection. Lymphocyte cultures from patients with the acquired immunodeficiency syndrome (AIDS) or pre-AIDS yielded virus rapidly, as indicated by high levels of reverse transcriptase (RT) activity in culture fluids. These viruses were able to establish a persistent infection in several T4-antigen-positive tumour cell-lines. In contrast, lymphocyte cultures from patients with mild or no symptoms yielded virus more slowly and the RT activity was low. Co-cultivation of slow/low-yielding lymphocytes with T4-positive tumour cell-lines showed no or only transient virus production. In 14 out of 23 cases virus could be detected by their fatal cytopathic effects on tumour cells. The relation between severity of illness and in-vitro replication potential of the viruses suggests that in the course of an infection selection may occur for HIV variants that replicate efficiently in T4 cells.

Leukocyte‐Cell Adhesion: A Molecular Process Fundamental in Leukocyte Physiology

Leukocyte-cell adhesion is a form of physical contact characterized by fast (firm) stickiness between the cells. To analyze the biology and molecular basis of this process, an adhesion-specific assay was developed: the phorbol ester-induced aggregation of human lymphocytes. This rapid and antigen-independent intercellular adhesion requires cellular metabolism, an intact cytoskeleton and extracellular divalent cations, and is mediated by preformed cell-surface proteins referred to as CAMs. Phorbol ester also induces aggregation of monocytes and granulocytes, as well as adhesion of T lymphocytes to either B cells or monocytes and of the leukocytes to vascular endothelial cells. By using the adhesion-specific assay and blocking monoclonal antibodies, several CAMs have been identified, namely the Leu-CAM family (CD11a-c/CD18) and ICAM-1 (CD54). The Leu-CAM family is composed of Leu-CAMa (CD11a/CD18), Leu-CAMb (CD11b/CD18) and Leu-CAMc (CD11c/CD18), three glycoprotein heterodimers made of a common beta-chain and distinct alpha-chains. ICAM-1 is an adhesive ligand for Leu-CAMa. Expression and use of the various CAMs is selective in different types of leukocytes. The Leu-CAMs have been purified and partially characterized. CD18, whose gene is on human chromosome 21, contains 5-6 N-linked complex-type oligosaccharides, and CD11 binds Ca++. Another adhesion pathway is mediated by CD2 and CD58. CD2, a glycoprotein selectively expressed by T cells, is a receptor for CD58, a cell-surface adhesive ligand with broad tissue distribution. Antibodies to the latter CAMs do not block the phorbol ester-induced lymphocyte aggregation. Adhesion is involved in a large variety of leukocyte functions. Anti-Leu-CAM antibodies block induction of IL-2 production and lymphocyte proliferation. Lymphocyte-mediated cytotoxicity is also inhibited. Endogenous NK and LAK cells use Leu-CAMs, ICAM-1 and CD2, and sometimes RGD receptors, to bind and kill tumor cells. Endogenous compounds such as H2O2 and LTB4 also induce Leu-CAM-dependent adhesion in monocytoid cells and granulocytes, respectively, and degranulation of the latter cells is enhanced by the adhesion process. Homologous CAMs have been identified in rabbit and mouse. In in vivo studies in the former species, anti-Leu-CAM antibodies block adhesion of leukocytes to vascular endothelium and thereby their migration into extravascular tissues. The antibodies thus inhibit granulocyte accumulation and plasma leakage in inflammatory lesions, and induce lympho- and granulocytosis, indicating that cell-adhesion contributes to the distribution of leukocytes in the body.(ABSTRACT TRUNCATED AT 400 WORDS)

ANTIBODY RESPONSE IN PRIMARY HUMAN IMMUNODEFICIENCY VIRUS INFECTION

The antibody response in 20 homosexual men with symptomatic primary HIV infection was studied with ten different antibody assays (enzyme-linked immunosorbent assays, indirect immunofluorescence assays, radioimmunoprecipitation [RIPA], and western blot). HIV antibodies were detectable by all the assays within 2 months after onset of illness. RIPA and western blot were more sensitive than the other assays--all serum samples obtained at 2 weeks and after were reactive. In all cases, the first serum sample reactive by RIPA precipitated gp160 whereas, by western blot, antibodies to p24 were first recognised. This study shows the necessity of including gp160 and p24 in any assay to detect early antibody response in primary HIV infection. 5 patients were studied by virus isolation. During the 2 first weeks after onset of symptoms, HIV was demonstrated in cell-free plasma in all cases and, in 4 cases, also in peripheral blood mononuclear cells. Samples obtained later contained demonstrable infectious virus in only 1 of 4 cases. Thus a phase of viraemia precedes the antibody response in symptomatic primary HIV infection.

Susceptibility to infection by the human immunodeficiency virus (HIV) correlates with T4 expression in a parental monocytoid cell line and its subclones

The monocytoid tumor cell line U-937 and five derived subclones were infected with the HTLV-IIIB isolate of the human immunodeficiency virus (HIV). Susceptibility to infection and sensitivity to the cytopathic effects correlated with the expression of the T4 antigen on the cell surface. On the basis of these characteristics the lines could be divided into three groups. Less than 10% T4 positive cells were present in the parental line and clone 4; hence productive infection could only be established after a long latency or with a high virus inoculum. These lines showed no or only marginal cytopathic effect. Clone 16 contained more than 95% T4 positive cells and was the most sensitive line to infection with HTLV-IIIB and its cytopathic effect. Cell death was so extensive following infection that no continuous virus producer line could ever be established from clone 16 cells. Cultures with intermediate T4 expression (50-70% T4+ cells) also had intermediate susceptibility to virus infection. Cytopathic changes, even if pronounced, could be overcome in the infected cultures by the addition of uninfected cells and, in each case, a producer line could be established. HTLV-IIIB infection of clone 16 cells could be blocked by preincubation with monoclonal anti-T4 antibodies indicating a close similarity between the HTLV-IIIB receptor on T4 positive T cells and monocytoid cells. The results thus show that T4 positive monocytoid cells can function as target cells for the HIV.

Human immunodeficiency virus infection of the brain: I. Virus isolation and detection of HIV specific antibodies in the cerebrospinal fluid of patients with varying clinical conditions

Isolation of the human immunodeficiency virus (HIV) has been attempted from the cerebrospinal fluid (CSF) of 29 subjects with varying severity of HIV infection. Virus could be isolated from patients in all stages of disease including patients with primary HIV infection and asymptomatic carriers. In the early stages of infection free virus was infrequently present in the CSF but could be isolated from the cells present in CSF. This suggests that HIV may reach the brain at a very early stage of infection and that initially there is little production of virus from infected cells. In the late stages of HIV infection, associated with increasing severity of immunodeficiency, free virus could readily be isolated from the CSF. With one exception, all of these patients had neurological and/or psychiatric symptoms, as compared to only 2 (of 13) subjects in the early stages of infection. All patients with HIV-specific antibodies in serum had antibodies also in CSF. Examined by a radioimmunoprecipitation assay, CSF was more often found to contain antibodies to the precursor (p55) of viral core proteins than the corresponding serum of the patients. We propose that immune disturbances have an essential pathogenic role in the neurological/psychiatric symptoms associated with HIV infection, possibly through allowing increased viral expression in the central nervous system.

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.

HIV-1 Infection of normal human macrophage cultures: Implication for silent infection

We have investigated the replicative capacity of 14 primary HIV-1 isolates in cultures of normal blood macrophages and PHA-stimulated peripheral blood mononuclear cells (PBMC). All viruses could infect normal macrophages as demonstrated by either reverse transcriptase (RT) activity, p24 antigen assay, or cocultivation with PBMC. One month after infection of macrophages virus could no longer be detected in the culture medium. The cells remained in this nonproductive state for another month. Virus could, however, be recovered by cocultivation with PHA-stimulated PBMC. Such macrophage-passaged virus induced pronounced cell killing with fragmentation and pyknosis and often replicated poorly in PBMC, in contrast to the original isolate. The results indicate that all primary HIV-1 isolates contain virus variants that can infect cells of the monocyte/macrophage lineage. Persistently infected, seemingly nonproducing cells, may serve as infectious reservoirs in the infected individual and spread infection to other susceptible cells over a long period of time. Moreover, the pronounced killing of PBMC by the macrophage-harbored virus may contribute to the deterioration of the immune system.

Improved tissue culture technique for production of poorly replicating human immunodeficiency virus strains

We present an improved culture technique for propagation of human immunodeficiency virus (HIV) strains that grow slowly, yield low reverse transcriptase activity and can be transmitted poorly, if at all, to cell lines. By using the Jurkat-tatIII cell line 29 (of 30) of these slow/low HIV strains, including one West African strain, could be shown to replicate over a period of several weeks.

Chromosomal, histopathological and cell surface marker studies on moloney virus induced lymphomas

Abstract Trisomy of chromosome 15 is a common feature in murine T-cell leukemias of chemical, viral and spontaneous origin. It is not present in all T-cell derived lymphomas, however. In order to study whether trisomy 15 was restricted to a specific T-cell subtype, diploid and chromosome 15 trisomic M-MuLV induced leukemias and derived in vitro lines were compared with regard to histology, karyotype, Thy-1,2 and Lyt-1, 2, 3 and 5 antigen expression. Histologically trisomic lymphomas showed a higher mitotic rate, higher peri-cellular reticulum content and less small cell infiltration than diploid tumors. It was not possible to distinguish trisomic from diploid lymphomas in blind tests based on these criterias, however. Trisomic and diploid lymphoma cells were indistinguishable in morphology. Three of nine lymphomas induced by M-MuLV inoculation were trisomic; two of them were thymomas. Transplantation to syngeneic recipients of the opposite sex and study of the sex chromosome marker was used to establish the origin of the enlarged organ. Upon subcutaneous transplantation, trisomic tumors and thymomas grew locally and were of donor cell type whereas splenic lymphomas caused spleen enlargement, as a rule, and were of donor or recipient type. Transplantation of bone marrow from mice infected with M-MuLV as newborns gave rise to lymphomas with the same sex as the donor and established the presence of preleukemic T-cells in the bone marrow of the donor. In vitro lymphoma lines expressed four different patterns of Lyt antigens: (a) 1.2 + , 2.2 + , 3.2 + , 5.1 + (4 lines); (b) 1.2 + , 2.2 − , 3.2 − , 5.1 + (5 lines); (c) 1.2 + , 2.2 + , 3.2 − , 5.1 + (7 lines); (d) individually distinct patterns (3 lines). Each group contained Thy-1.2 − and Thy-1.2 + lines. The trisomic tumors were mostly in group (a), all of them were Thy-1.2 positive. Diploid tumors were randomly distributed largely between groups (b) and (c). Two of three tumors in group (d) were aneuploid (not trisomic for 15). In spite of the differences observed between trisomic and diploid lymphomas, no specific T-cell subset could be assigned to the two groups. This is in line with the finding of trisomy 15 in some B-cell lymphomas suggesting that this anomaly may contribute to the neoplastic change in a variety of lymphocyte subsets, representing different lineages and steps of differentiation.