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The activity of natural killer cells was found to be deficient in 10 of 12 males with X-linked lymphoproliferative syndrome, a life-threatening proliferation of lymphocytes after infection by Epstein-Barr virus. The activity levels of natural killer cells from affected males were increased after treatment with interferon in vitro, but normal levels of killing were not obtained. Deficient activity of killer cells in individuals with immunodeficiency and chronic infection by Epstein-Barr virus may contribute to the development of lymphoproliferative disorders.

Abstract. The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A)RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.

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A B S T R A C T The X-linked lymphoproliferative syndrome is characterized by immunodeficiency to Epstein-Barr virus (EBV) manifested by severe or fatal infectious mononucleosis and acquired immunodeficiency. We studied immune responses in six males of a well-characterized kindred with the X-linked lymphoproliferative syndrome. Two males were studied before and during acute fatal EBV infection. Both individuals demonstrated normal cellular and humoral immunity before EBV infection. During acute EBV infection, both individuals developed vigorous cytotoxic cellular responses against EBV-infected and -iipinfected target cells. Anomalous killer and natural killer T cell activity was demonstrated against a va'-riety of lymphoid cell lines, autologous fibroblasts and autologous hepatocytes. Effector cells responsible for anomalous killing reacted with a pan-T cell monoclonal antibody, and belonged to the OKT.8 T cell subset. Death in each case was caused by liver failure, but one patient developed extensive liver necrosis, whereas the other developed a massive infiltration of the liver with EBV-infected immunoblasts after aggressive immunosuppressive therapy. Immunological studies were performed on four males who had survived EBV infection years previously. They demonstrated global cellular immune defects with deficiencies of lymphocyte proliferative responses to mitogens and antigens, humoral immune deficiencies, abnormalities of regulatory T cell subsets and deficient natural killer cell activity. We propose that an aberrant immune response triggered by acute EBV infection results in unregulated anomalous killer and natural killer cell activity against EBV infected and uninfected cells. These studies suggest that global immune defects appearing in males with X-linked lymphopro-

The X-linked lymphoproliferative syndrome is triggered by Epstein-Barr virus infection and results in fatal mononucleosis, immunodeficiency, and lymphoproliferative disorders. This study shows that the mutation responsible for X-linked lymphoproliferative syndrome is genetically linked to a restriction fragment length polymorphism detected with the DXS42 probe (from Xq24-q27). The most likely recombination frequency between the loci is 4%, and the associated logarithm of the odds is 5.26. Haplotype analysis using flanking restriction fragment length polymorphism markers indicates that the locus for X-linked lymphoproliferative syndrome is distal to probe DXS42 but proximal to probe DXS99 (from Xq26-q27). It is now possible to predict which members of a family with X-linked lymphoproliferative syndrome are carrier females and to diagnose the syndrome prenatally.Epstein-Barr virus (EBV)-associated diseases-infectious mononucleosis, nasopharyngeal carcinoma, and Burkitt lymphoma-cause considerable morbidity and mortality. After EBV infection B lymphocytes are efficiently transformed into persistently proliferating lymphoblastoid cells. These cells can be killed by other proliferating cells that are evoked in an immune response. Infection of children is usually asymptomatic, but nonimmune adults develop infectious mononucleosis. EBV-induced lymphoproliferation is fatal in some new world primate species and in a small number of human subjects with both inherited and acquired immunodeficiencies. One such defect results from a mutation of the X chromosome.This X-linked lymphoproliferative (XLP) syndrome mutation in some way prevents the immune system of an affected male from making an appropriate response to EBV infection (1-4). Before EBV infection occurs, males with an XLP mutation have normal cellular and humoral immune responses (5) and respond normally to bacterial and viral infections other than EBV. After EBV infection, about 75% of males with XLP develop fatal infectious mononucleosis, with liver destruction often the immediate cause of death. Boys with XLP who survive EBV infection have defects in humoral and cellular immunity and a high incidence of lymphoma (4-6). The interaction between EBV and B lymphocytes appears normal in affected males, but EBV-induced activation of anomalous killer T cells may cause the lymphoreticular cytopathology that leads to symptoms (5).Restriction fragment length polymorphism (RFLP) markers are being used to map genetic disease loci and to construct the human genetic map (7). In this report we describe the use of X chromosome RFLPs to study a family with XLP. The discovery of RFLPs flanking the XLP locus will enable diagnosis prior to infection and may prove an important step toward understanding XLP at the molecular level. [a-32P]dATP. The blots were washed in 0.08% NaCl/0.1% NaDodSO4/0.08% Tris base/ 0.02% H3PO4 at 60°C and autoradiographed with an intensifying screen at -70°C.RFLP probes used and their locations are as follows:

Rat skeletal muscle cells and a cloned myogenic cell line synthesize and secrete in culture a molecule that is immunologically and biologically indistinguishable from the active form of nerve growth factor (NGF) from mouse submandibular gland. This protein can be detected in medium conditioned by muscle cells both before and after fusion and in the soluble fraction of muscle cell homogenates. Chromatographic data also reveal that the molecular properties of muscle cel NGF differ from those of the growth factor purified from mouse submandibular glands. Musce cell NGF has a molecular weight between 140,000 and 160,000, whereas purified mouse gland NGF has a molecular weight of G-200 and G-75, from Pharmacia; D-arabinofuranosylcytosine (Ara C) and cycloheximide, from Calbiochem; bovine serum albumin (3X crystallized), trypsin, and horse heart ferricytochrome c, from Sigma; 3H20 and NaI25I from New England Nuclear; and human IgG from Pentex. Rat tail collagen was prepared by the procedures of Ehrmann and Gey (7). Calf skin collagen without preservative was purchased from Worthington. NGF was isolated and purified from male mouse submandibular glands by modifications (1) of the method of Bocchini and Angeletti (8). All preparations were electrophoretically homogeneous as previously described (1).Antisera to NGF were prepared in rabbits (1). IgG was purified by diluting serum with an equal volume of 0.15 M NaCl in 10 mM sodium phosphate, pH 7.4. This solution was brought to 50% saturation with ammonium sulfate, and the precipitate was redissolved in and dialyzed against 10 mM sodium phosphate/15 mM NaCl at pH 7.5. The solution was applied to a column of Abbreviations: NGF, nerve growth factor; Ara C, D-arabinofuranosylcytosine.

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.A sensitive high-resolution detection of human immunodeficiency virus (HIV) MATERIALS AND METHODSCells and Cell Preparation. Normal T lymphocytes were cultured for 3-5 days with interleukin 2 and phytohemagglutinin and infected with HIV-1 (strain IIIB) at a low multiplicity of infection. At daily intervals, samples were taken for in situ hybridization. Cells were washed in isotonic phosphate-buffered saline (PBS) and applied to multi-well serologic slides (5-mm wells, Cel-line) at 250 x 106 cells per ml (2).The following reagents were obtained through the AIDS Research and Reference Reagents Program: C816645 (8) and 8E5/LAV cells (9). The 8E5/LAV cell line contains (9) a single viral genome with a defective pol gene. Cytogenetic preparations of8E5/LAV cells were prepared and hybridized as described (6).In Situ Hybridization. Hybridization was performed essentially as described and was the same for biotin-or digoxigenin-labeled probes (6,7). The probes were labeled by nicktranslation as described (2). After fixation in 4% (wt/vol) paraformaldehyde/PBS for 5 min, slides were dehydrated through a graded ethanol series and air-dried. The 5420The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.