Macrophages are required for influenza virus infection of human lymphocytes.

Monocyte and lymphocyte surface-expressed viral antigens have been demonstrated after exposure of unseparated human mononuclear leukocytes to influenza virus in vitro. The current studies, using [35S]methionine pulse-labeled purified preparations of virus-exposed macrophages, depleted of lymphocytes, demonstrate that the presence of these viral proteins does represent new synthesis. However, purified lymphocytes, depleted of monocytes-macrophages and exposed to influenza virus, showed no detectable viral protein synthesis. In further experiments, unseparated mononuclear leukocytes were exposed to virus and subsequently separated by countercurrent centrifugal elutriation. Both macrophages and lymphocytes were then shown to synthesize influenza proteins. Cell-free control or influenza virus-infected macrophage-derived supernatant fluids did not facilitate influenza virus infection of the lymphocytes. The data suggest that macrophages are required for influenza virus infection of human lymphocytes, and raise the possibility that macrophage facilitation of an abortive infection of lymphocytes plays a role in the generation of effective immunity to viral antigens.

Potential environmental and host participants in the early white matter lesion of adreno-leukodystrophy: morphologic evidence for CD8 cytotoxic T cells, cytolysis of oligodendrocytes, and CD1-mediated lipid antigen presentation.

The 2 most common forms of X-linked adreno-leukodystrophy (ALD) are the juvenile or childhood cerebral form with inflammatory demyelination and the adult adrenomyeloneuropathy (AMN) involving spinal cord tracts without significant inflammation. Modifier genes or environmental factors may contribute to the phenotypic variability. We performed immunohistochemical, an in situ polymerase chain reaction, and TUNEL analyses to identify several viruses, lymphocyte subpopulations, apoptotic cells, and effector molecules, focusing on morphologically normal white matter, dysmyelinative and acute demyelinative lesions. No distinguishing viral antigens were detected. Most lymphocytes were CD8 cytotoxic T cells (CTLs) with the alpha/beta TCR, and they infiltrated morphologically unaffected white matter. Only a few oligodendrocytes were immunoreactive for caspase-3. MHC class II- and TGF-beta-positive microglia were present. CD44, which can mediate MHC-unrestricted target cell death, was seen on many lymphocytes and white matter elements. CD1 molecules, which play major roles in MHC-unrestricted lipid antigen presentation, were noted. Our data indicate that unconventional CD8 CTLs are operative in the early stages of dysmyelination/demyelination and that cytolysis of oligodendrocytes, rather than apoptosis, appears to be the major mode of oligodendrocytic death. The presentation of lipid antigens may be a key pathogenetic element in ALD and AMN-ALD.

Role of the monocyte-macrophage in influenza virus infection of lymphocytes: implications for HIV infection.

Knowledge of the pathogenesis of viruses which are less virulent than human immunodeficiency virus (HIV) may provide valuable insights into the pathogenesis of HIV infection. Influenza virus, an enveloped RNA virus, infects monocyte-macrophages, although the infection is brief and abortive. Isolated purified lymphocytes are completely resistant to infection. In contrast, mixtures of lymphocytes and macrophages can synthesize all virus proteins. Infection requires physical association of monocyte-macrophages and lymphocytes in "clusters." These studies with influenza virus suggest that the pathogenesis of virus infections in mixed cell cultures may be very different from that observed in purified cell populations, and they suggest that similar studies should be performed with HIV.

The HHV6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) and multiple sclerosis (MS) are demyelinative diseases of the central nervous system (CNS). PML occurs mostly in individuals with AIDS-impaired immunity and is thought to be caused by JC polyoma virus (JCV). In MS a neurotrophic virus trigger is suspected, but the precise etiology remains unknown. Human herpesvirus 6 (HHV6) is a ubiquitous, commensal and usually benign beta-herpesvirus. Some researchers have found evidence for HHV6 infection in MS plaques and sera. We recently demonstrated a high frequency of cells containing HHV6 genome in PML lesions, as well as co-infection of oligodendrocytes by JCV and HHV6. This suggests that HHV6 may be a co-factor in the etiology of PML, and raises questions about its role in other demyelinative diseases. OBJECTIVES: To determine the prevalence and cellular localization of HHV6, JCV and HIV-1 infected cells in PML, MS, AIDS and control CNS tissues, and their potential relationship with disease. STUDY DESIGN: An unconventional, sensitive two-step in situ polymerase chain reaction (ISPCR) procedure was used to amplify and detect HHV6, JCV and HIV-1 genomic DNAs in formalin fixed, paraffin-embedded archival CNS tissues. HHV6, JCV and HIV-1 gene expression was detected by ICC for HHV6 p41 and gp101, JCV large T, and HIV-1 p24 gag and NEF proteins. RESULTS: A high frequency of HHV6 genome was consistently detected in both PML and MS white matter lesional cells; a peri-lesional concentration was notable. HHV6 was found mainly in oligodendrocytes, but neurons were also infected. HHV6 was present in larger amounts than JCV in PML lesions, while more HIV-1 than HHV6 was present in AIDS. Variable amounts of HHV6 genome were detected in normal, AIDS and other control brains; the frequency of infected cells tended to increase with patient age. CONCLUSIONS: High concentrations of HHV6 genome in association with PML and MS lesions, open the possibility that HHV6 activation may play a role in the pathogenesis of these demyelinative diseases.

Use of FITC-labeled influenza virus and flow cytometry to assess binding and internalization of virus by monocytes-macrophages and lymphocytes.

The binding of influenza virus to the surface of cells and the internalization of virus particles by all or a subset of cells are key points in the pathogenesis of viral infection. The current studies established a method for discrimination of surface-bound from internalized influenza virus. Fluorescein isothiocyanate (FITC) was attached to the viral hemagglutinin and neuroaminidase proteins; the fluorescent virus retained infectivity. A flow cytometric technique was then adapted for study of virus-cell interactions, with addition of ethidium bromide to quench green fluorescence associated with FITC-labeled virus that was cell-bound but remained external. Ethidium bromide was excluded by intact cell membranes, and internalized virions retained green fluorescence. Cells could be examined by fluorescence microscopy or flow cytometry, with flow cytometry allowing rapid, kinetic assessment of large numbers of cells and subsets of virus-exposed cells. The data showed that, whereas a majority of both monocytes-macrophages and lymphocytes bound influenza virus, a large percentage of monocytes-macrophages but only a very small percentage of lymphocytes internalized the virus. This procedure provides a simple and effective method to distinguish surface-bound from internalized influenza virus, and allows precise kinetic analyses on large numbers of cells.

Human herpesvirus 6.

The development of techniques for the culture of lymphoid cells and the isolation of viruses that infect these cells led to the discovery of human herpesvirus (HHV) 6 in 1986. At the time, HHV-6 was the first new human herpesvirus to be discovered in roughly a quarter of a century, and its isolation marked the beginning of an era of discovery in herpesvirology, with the identification of HHV-7 and HHV-8 (Kaposi's sarcoma-associated herpesvirus) during the following decade. Like most human herpesviruses, HHV-6 is ubiquitous and capable of establishing a lifelong, latent infection of its host. HHV-6 is particularly efficient at infecting infants and young children, and primary infection with the virus is associated with roseola infantum (exanthem subitum) and, most commonly, an undifferentiated febrile illness. Viral reactivation in the immunocompromised host has been linked to a variety of diseases, including encephalitis, and HHV-6 has been tentatively associated with multiple sclerosis. This article discusses the major properties of HHV-6, its association with human disease, and the pathobiological significance of viral reactivation.

Angiotropic lymphoma manifested by fever and painful swollen legs.

In summary, angiotropic lymphoma is generally of B-cell origin and can be manifested by isolated cutaneous lesions. More commonly, however, the disease involves the CNS. Angiotropic lymphoma should be included in the differential diagnosis of patients with clinical features of panniculitis or diffuse multisystem vasculitis.

Early identification and retrieval or deletion of human lymphocyte subpopulations responding to influenza virus or respiratory syncytial virus challenge.

Differences in immune responses of human mononuclear leukocytes (MNL) have been demonstrated following exposure in vitro to influenza virus or respiratory syncytial virus (RSV). In the current studies, we sought to identify early differences in reactive subpopulations that emerge from within the heterogeneous resting MNL pool after challenge. MNL were sham-exposed or exposed to influenza virus or RSV, separated, and retrieved by countercurrent centrifugal elutriation after 3 d. Exposure to influenza virus caused a relative decline in the number of large MNL, but an increase in small lymphocytes. Large cells that remained included primitive lymphoblasts, rare plasma cells, and typical lymphocytes of progressively greater volume. Exposure to RSV increased the number of large MNL, but diminished the number of small lymphocytes. The subpopulation of large cells consisted of atypical and large granular lymphocytes. Furthermore, deletion of the latter large, reactive lymphocytes led to abrogation of an RSV-specific proliferative response upon subsequent challenge. Thus, the specific and different subpopulations reactive after infectious virus challenge could be identified, retrieved, and manipulated without dependence on a priori, phenotypic markers.

Human herpesvirus 6-meningoencephalitis in an HIV patient with progressive multifocal leukoencephalopathy.

Human herpesvirus 6 (HHV6) has been reported as a rare cause of meningoencephalitis and leukoencephalitis. We present an HIV-infected patient with lesions of progressive multifocal leukoencephalopathy (PML), but also meningoencephalitis apparently due to HHV6. Immunohistochemistry for HHV6 antigens and in situ polymerase chain reaction for HHV6 genome showed many positive lymphocytes and microglia in the meningeal and cortical lesions. More importantly, dead and dying neurons were conspicuous; some were undergoing neuronophagia and some displayed evidence of HHV6 infection. A pathogenic role for this almost universal, and usually commensal, virus in inflammatory brain lesions and PML is briefly discussed.

Association of human herpesvirus 6 with the demyelinative lesions of progressive multifocal leukoencephalopathy.

Progressive Multifocal Leukoencephalopathy (PML) is a primary demyelinating disease of the central nervous system occurring almost exclusively in individuals with impaired cell-mediated immunity. The JC polyoma virus has been accepted as the etiologic agent ofPML. Using a two-step in-situ polymerase chain reaction procedure to amplify and detect genomic DNA of human herpesvirus-6 (HHV6) in formalin-fixed paraffin-embedded archival brain tissues, a high frequency of infected cells was consistently detected in PML white matter both within and surrounding demyelinative lesions and HHV6 genome was found mainly within oligodendrocytes. Lesser amounts of HHV6 genome were detected in most normal, AIDS, and other neurological disease control tissues. Immunocytochemistry for HHV6 antigens showed actively infected nuclei of swollen oligodendrocytic morphology only within the demyelinative lesions of PML but not in adjacent uninvolved tissue. In addition, no HHV6 antigens were detectable in control tissues including brains of individuals with HIV-1 encephalopathy but without PML. Double immunohistochemical staining for JC virus large T antigen and HHV6 antigens demonstrated co-labeling of many swollen intralesional oligodendrocytes in the PML cases. The evidence suggests that HHV6 activation in conjunction with JC virus infection is associated with the demyelinative lesions of PML.