Essential role of mitochondrial antiviral signaling, IFN regulatory factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-mediated IFN-beta response and control of bacterial replication in human endothelial cells.

Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-beta). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-beta, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-beta induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-beta treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

Change in the structure of Shope papilloma virus-induced arginase associated with mutation of the virus.

The change in the state of the virus-induced enzyme associated with a mutation in the virus provides additional evidence that the enzyme is synthesized from virus rather than rabbit genetic information. This change in structure results in differences in stability of polymerization, degree of optical rotary dispersion (ORD) specific rotation, change in elution characteristics from carboxymethyl cellulose, and a reduction in specific activity of the arginase. Liver arginase differs markedly in ORD characteristics from the virus-induced enzyme. In contrast to the virus-induced enzyme, it showed no negative Cotton effect at 233 nm until it was activated with manganese. Manganese had no influence on the ORD spectrum of virus-induced arginase. In addition, liver arginase is denatured by 4 M urea, while the virus-induced enzyme requires 10 M urea for denaturation.

B cells are required for induction of T cell abnormalities in a murine retrovirus-induced immunodeficiency syndrome.

The role of B cells in induction of phenotypic and functional abnormalities of T cells in a murine retrovirus-induced immunodeficiency syndrome, MAIDS, was evaluated in mice depleted of mature B cells from birth with anti-IgM antibodies (mu-suppressed) and infected at 4 wk of age. Multicolor FACS analyses of CD4+ T cell subsets showed that development of phenotypic abnormalities of these cells at 9 wk after infection was completely inhibited by mu-suppression. Furthermore, induction of impaired proliferative responses to Con A and alloantigens and CTL responses to alloantigens was fully blocked in antibody-treated animals. The extent of virus replication was comparable in spleens of untreated and mu-suppressed mice. Retroviral induction of T cell dysfunction in MAIDS is thus dependent on the presence of B cells, and high level virus expression in mice without B cells has little or no effect on T cell function.

Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction.

Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.

A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T.

We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Detection of papillomavirus DNA in human semen.

Human papillomavirus DNA has been detected in the semen of three patients, two of whom have severe chronic wart disease. These data support the contention that sexual transmission of human papillomavirus DNA could occur via semen, a possibility suggested by epidemiological data on the sexual transmission of human papillomavirus.

Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).

A retrovirus belonging to the family of recently discovered human T-cell leukemia viruses (HTLV), but clearly distinct from each previous isolate, has been isolated from a Caucasian patient with signs and symptoms that often precede the acquired immune deficiency syndrome (AIDS). This virus is a typical type-C RNA tumor virus, buds from the cell membrane, prefers magnesium for reverse transcriptase activity, and has an internal antigen (p25) similar to HTLV p24. Antibodies from serum of this patient react with proteins from viruses of the HTLV-I subgroup, but type-specific antisera to HTLV-I do not precipitate proteins of the new isolate. The virus from this patient has been transmitted into cord blood lymphocytes, and the virus produced by these cells is similar to the original isolate. From these studies it is concluded that this virus as well as the previous HTLV isolates belong to a general family of T-lymphotropic retroviruses that are horizontally transmitted in humans and may be involved in several pathological syndromes, including AIDS.

Proviral DNA of a retrovirus, human T-cell leukemia virus, in two patients with AIDS.

The acquired immune deficiency syndrome (AIDS) is characterized by T-lymphocyte dysfunction and is frequently accompanied by opportunistic infections and Kaposi's sarcoma. Human T-cell leukemia virus (HTLV) is associated with T-cell malignancies and can transform T lymphocytes in vitro. In an attempt to find evidence of HTLV infection in patients with AIDS, DNA from samples of peripheral blood lymphocytes from 33 AIDS patients was analyzed by Southern blot-hybridization with a radiolabeled cloned HTLV DNA probe. Analysis of DNA from both the fresh (uncultured) lymphocytes and from T cells cultured with T-cell growth factor revealed the presence of integrated HTLV proviral sequences in lymphocytes from two of the patients, both of whom had antibody to HTLV. The proviral sequences could not be detected in blood samples obtained from these individuals at a later date, consistent with the possibility that the population of infected cells had become depleted.

Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS).

Several isolates of a human type-C retrovirus belonging to one group, known as human T-cell leukemia virus (HTLV), have previously been obtained from patients with adult T-cell leukemia or lymphoma. The T-cell tropism of HTLV and its prevalence in the Caribbean basin prompted a search for it in patients with the epidemic T-cell immune deficiency disorder known as AIDS. Peripheral blood lymphocytes from one patient in the United States and two in France were cultured with T-cell growth factor (TCGF) an shown to express HTLV antigens. Virus from the U.S. patient was isolated and characterized and shown to be related to HTLV subgroup I. The virus was also transmitted into normal human T cells from umbilical cord blood of a newborn. Whether or not HTLV-I or other retroviruses of this family with T-cell tropism cause AIDS, it is possible that patients from whom the virus can be isolated can also transmit it to others. If the target cell of AIDS is the mature T cell as suspected, the methods used in these studies may prove useful for the long-term growth of these cells and for the identification of antigens specific for the etiological agent of AIDS.

Antibodies to cell membrane antigens associated with human T-cell leukemia virus in patients with AIDS.

The acquired immune deficiency syndrome (AIDS), which has recently occurred at increasing rates in homosexual men, intravenous drug users, and others, is characterized by the development of Kaposi's sarcoma and several opportunistic infections including pneumonia caused by Pneumocystis carinii. Serum samples from patients with AIDS and from matched and unmatched control subjects were examined for the presence of antibodies to cell membrane antigens associated with human T-cell leukemia virus. Nineteen of 75 of the AIDS patients had antibodies directed to surface antigens of Hut 102, a reference T lymphoid cell line infected with leukemia virus, as did two of the 336 control subjects.

Physiologic concentrations of normal human plasma lipoproteins inhibit the immortalization of peripheral B lymphocytes by the Epstein-Barr virus.

Epstein-Barr virus (EBV)-induced immortalization of adult human B lymphocytes is suppressed by physiologic concentrations of human plasma lipoproteins. Several inhibitory mechanisms appear to be operative. First, low density lipoproteins (LDL) directly reduce the ability of EBV to transform human B cells. Second, LDL as well as intermediate and very low density lipoproteins modulate early inductive events rendering the B cell refractory to transforming signals from EBV. Third, LDL also selectively inhibit an EBV-inducible step that occurs within 24 h after transformation. Finally, very low density lipoproteins can abrogate the ongoing, cellular proliferation of EBV-transformed, established B cell lines. The plasma lipoproteins may therefore prevent the emergence of EBV-transformed malignant B cell clones in vivo. Conceivably, on this basis, environmental and genetic influences on plasma lipoprotein concentrations may affect the global distribution of Burkitt's lymphoma, a lymphoid malignancy putatively caused by EBV.

Host-clonal interactions in the generation of proviral gene deletion variants.

A nonproducer clone (clone A1) (from a retrovirus-infected guinea pig fibrosarcoma) has been described that under conditions of in vivo immunologic selection forms variants that lack the proviral gene. One trivial explanation for the apparent loss of the provirus from clone A1 is that clone A1 did not originate from a single cell. For evaluation of this possibility, subclones were derived from clone A1 and tested for tumor recurrence in nonimmune and virus-immune animals. Each of four subclones contained the A1 provirus and exhibited specific viral interference; tumor recurrences formed from each of these four subclones lacked the clone A1 provirus. Possible, when heterogeneous populations of retrovirus-infected cells are injected into nonimmune animals, some clones will elicit immunologic responses to retroviral antigens and subject other clones in the population to immunologic selective pressures. For testing this concept, clone A1 was injected in admixture with a producer clone (clone A4) into nonimmune Sewall Wright strain 2 guinea pigs. Tumors formed in nonimmune guinea pigs inoculated with clone A1 in admixture with clone A4 were shown to lack a detectable clone A1 provirus. The results supported the concept that a somatic mutational event (deletion of the proviral gene) occurs during growth of clone A1. When heterogeneous populations of retrovirus-infected cells are injected into animals, host-clonal interactions may occur leading to outgrowth of proviral gene deletion variants. These results supported the notion that interactions between tumor clones and the host can change the dominant clonal type of the tumor and provide a genetic basis for this change.

In vivo immunologic selection of proviral gene deletion variants from a nonproducer clone.

The mechanism by which tumors recur at sites of injection of retrovirus-infected fibrosarcoma cell lines was investigated. Previously, it was established that tumor recurrences reflect outgrowth of rare cells that lack viral antigens and are susceptible to superinfection with the homologous retrovirus. In the present study clones isolated from a retrovirus-infected cell line were evaluated as precursors for tumor recurrence. Under conditions of in vivo immunologic selection, a clone that contained a single abbreviated copy of the provirus formed variants that lacked the proviral gene. Tumor variants lacking the proviral gene grew progressively in both nonimmune and virus-immune male Sewall Wright strain 2 guinea pigs. Tumor recurrence could be prevented by superinfection of the virus-infected fibrosarcoma cell line or by superinfection of the precursor for tumor recurrence. Cell lines infected with retroviruses varied in frequency of tumor recurrence formation. This model may be useful in analyzing gene deletion as a mechanism of tumor escape from host immunologic attack.

Immunologic relatedness of papillomaviruses from different species.

An antiserum prepared by immunization of a rabbit with sodium dodecyl sulfate-disrupted virions from a pool of plantar warts was cross-reactive with virus-positive papillomas of other animal species by both indirect immunofluorescence tests on frozen sections of wart tissues and peroxidase-antiperoxidase tests of sections of Formalin-fixed tissues. The antiserum stained plantar warts, common warts, and skin lesions of epidermodysplasia verruciformis, all from humans; bovine fibropapilloma, experimentally produced with bovine types 1 and 2; and transmissible canine oral papillomas. The staining was localized to nuclei of the upper granular layers of the peithelium and was similar in distribution to the pattern produced by antiserum specifically prepared against that papillomavirus. The antiserum did not stain virus-negative warts, or cells infected with simlan virus 40, human polyomavirus BK, and murine polyomavirus. These data suggested that papillomaviruses share a common internal antigen unrelated to a similar antigen described previously for the polyomaviruses (which include simian virus 40 and polyomavirus subgroups).

Inefficient humoral immune response of lymphoma-prone wild mice to persistent leukemia virus infection.

Adult wild mice (LC) from a natural colony with a high incidence of spontaneous lymphomas had free infectious virus in their seara (average 10(3.5) infectious units/ml) and parenchymal organs (average 10(5.2) infectious units/g tissue). They did not have detectable levels of free virus-specific antibodies that could be demonstrated by virus neutralization or immunofluorescence at higher than a 1:10 dilution. Only 5 of 28 animals had free antibodies detectable by radioimmunoprecipitation assay, and tissues of 4 mice also had nondetectable levels of virus determined by infectivity assay. Formalized vaccine from the indigenous virus did not induce production of virus-neutralizing antibodies or protect against naturally occurring disease. The animals with persistent leukemia virus infection, however, elicited good humoral immune responses to virus-unrelated antigen.

Virus isolation test for feline leukemia virus.

A method was developed for detecting feline leukemia virus (FeLV) infection in cats by coculturing separated blood lymphocytes and platelets with clone 81 cells carrying a murine sarcoma genome but free of leukemia virus (S+L-). FeLV caused cell transformation that was easily visible 3-12 days after infection. This method is simple, sensitive, and easily read; it has the advantage of directly detecting infectious FeLV rather than viral antigens.

Complete, circular papovavirus genomes in the cells of hamsters exposed to a horizontally transmitted lymphomagenic agent.

Contagious lymphomas were produced in a colony of Syrian golden hamsters by an unknown agent that also caused fatal ulcerative bowel disease (UBD) lesions prior to lymphoma development. A low percentage of these animals developed epitheliomas of the skin independently of the UBD or lymphomas. Previous work has shown that the epitheliomas contain numerous hamster papovavirus (HaPV) particles, whereas lymphomas do not. Cells from both kinds of tumors do contain HaPV DNA sequences, however. In this study, Southern blot hybridization showed that complete, circular HaPV genomes were present in these cells. Complete, circular HaPV genomes also were found in the cells of animals with UBD. Electron microscopy revealed the presence of HaPV particles in UBD lesions. These results, together with previous data, indicate that in the hamster, lymphomas contain complete, circular papovavirus genomes in the absence of virus particles, whereas epitheliomas and UBD lesions contain these genomes in the presence of virus particles.

Immunocytochemical distribution of mouse mammary tumor virus antigens in BALB/cfC3H mammary epithelium.

The distribution of mouse mammary tumor virus (MuMTV) antigens was studied in normal, preneoplastic, and neoplastic mammary epithelia from female BALB/cfC3H mice with the use of a polyvalent anti-MuMTV serum and indirect immunoperoxidase techniques. The MuMTV antigens were on the apical surface or in focal cytoplasmic aggregates or diffused throughout the infected cells. As much as 70% of all cells in adenocarcinomas and as much as 100% of all cells in preneoplastic hyperplastic alveolar nodules contained MMTV antigens. Comparable percentages of cells from mammary glands of multiparous mice were MuMTV-positive. Some mammary tissues of nullparous and primiparous mice did not contain detectable MuMTV antigen. The MuMTV antigen-containing cells in lactating mammary glands tended to be in discrete lobuloalveolar clusters surrounded by antigen-negative alveoli. The percentage of MuMTV-positive cells in a given gland was proportional to the amount of virus found in the animal's milk.

In vivo antigenic modification of tumor cells. II. Distribution of virus in sarcoma-bearing mice.

Murine leukemia viruses were previously demonstrated to be able to infect efficiently non-virus-expressing tumors in vivo. In the present study the infectivity and tissue distribution of Friend murine leukemia virus (F-MuLV) in normal and tumor-bearing C57BL/6J (B6) mice were examined. Two syngeneic fibrosarcoma-inducing cell lines were used: Cells from a 3-methylcholanthrene-induced fibrosarcoma syngeneic to B6 mice (MCA-FS) and cells from a Harvey murine sarcoma virus-transformed, nonproducer sarcoma syngeneic to B6 mice (H-NP) were described in the preceding study. Both cell lines lacked ecotropic viral expression. F-MuLV produced in vitro was rarely able to infect normal adult B6 tissue in vivo and lacked pathogenic potential. Adult animals receiving F-MuLV remained clinically normal during 20 months of follow-up and had no detectable viremia, although some had persistently infected thymuses and long bones. In animals receiving a single dose of F-MuLV given to superinfect either the MCA-FS or the H-NP induced tumors, virion antigens were found only in tumor tissue and not in the normal host organs studied. Infectious virus was abundant in tumors; occasionally, it was found in thymuses and long bones of animals bearing superinfected H-NP tumors but rarely in other organs. Localization of F-MuLV in MCA-FS tumors appeared to be more selective with rare contamination of host organs. The presence of a rescuable sarcoma genome in H-NP may explain the discrepancy between MCA-FS and H-NP tumors. The possibility of increasing the efficiency and selectivity of infection as well as the therapeutic application of this technique are discussed.