Peritoneal HPV-DNA test in cervical cancer (PIONEER study): A proof of concept.

The aim of this study was to investigate the prevalence of peritoneal human papillomavirus (HPV) infection in different clinical cervical cancer (CC) settings, and its association with potential clinical and/or histological factors. This is a single-center, prospective, observational study. Consecutive patients with newly diagnosed or recurrent/persistent CC, between March 2019 and April 2020, were included. A group of patients undergoing surgery for benign gynecological conditions was included as control group. All patients underwent HPV-DNA test in the cervix and in the peritoneal cavity simultaneously at time of surgery. Two-hundred seventy-two patients had cervical and peritoneal HPV test analyzed. Cervical and peritoneal HPV positivity (PHP) was found in 235 (88.0%) and 78 (28.7%) patients, respectively; the prevalence of PHP was 17.7% in early stage, 28.8% in locally advanced cervical cancer (LACC) and 46.6% in the metastatic/persistent/recurrent setting (P = .001). No control patient was found to have peritoneal HPV infection. Higher frequency of PHP was documented in patients with larger tumor size (P = .003), presence of cervical HPV 16/18 genotypes (P < .001), higher number of cervical high-risk (HR)-HPV per patient (P = .018) and peritoneal carcinomatosis (P < .001). Multivariate analysis demonstrated that lack of preoperative cervical conization in early stages (P = .030), while higher International Federation of Gynecology and Obstetrics (FIGO) stage (P = .021) and presence of cervical HPV 16/18 (P = .001) in LACC, was associated with PHP. This is a proof-of-concept study. A number of potential clinical implications, including prognosis, could be obtained by further studies.

Towards a mechanistic understanding of the synergistic response induced by bivalent Marek's disease vaccines to prevent lymphomas.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. METHODS: Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. RESULTS: The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. CONCLUSION: Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

Peritoneal Cells Mediate Immune Responses and Cross-Protection Against Influenza A Virus.

Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets. However, the responses of peritoneal cells to influenza A virus have not been investigated. Here we show that intraperitoneal inoculation with A/WSN/1933 (H1N1) virus induced virus-reactive IgG production in the peritoneal cavity in mice. The infection resulted in substantial but transient B cell and macrophage depletion along with massive neutrophil infiltration, but virus growth was not detected. Influenza A viruses bound to α-2,6-linked sialic acids of B cells and macrophages and induced apoptotic death of peritoneal cavity cells. However, re-infection with A/WSN/1933 virus did not have adverse effects on immune cells most likely because of the neutralizing antibodies produced in response to the first exposure. Infection of BALB/c mice with A/WSN/1933 induced cross-protection against an otherwise lethal intraperitoneal dose of A/Hongkong/4801/2014 (H3N2) virus. This information suggests that immunological responses in the peritoneal cavity can induce effective defense against future virus infection. Considering the unexpected potent immunoregulatory activity of the peritoneal cells against influenza viruses, we suggest that comparative studies on various immune reactions after infection through different routes may contribute to better selection of vaccination routes in development of efficacious influenza vaccines.

LXR Alpha Restricts Gammaherpesvirus Reactivation from Latently Infected Peritoneal Cells.

Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here, we demonstrate that the liver X receptor alpha isoform (LXRα) restricts gammaherpesvirus reactivation in an anatomic-site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (α and ß) leads to an increase in fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRα-deficient hosts, indicating that the antiviral effects of LXRα are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8+ T cells and interferon (IFN) signaling, remained intact in the absence of LXRα. Remarkably, using a murine gammaherpesvirus 68 (MHV68) reporter virus, we discovered that LXRα expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRα-/- mice exhibit reduced latency within the peritoneal B cell compartment and elevated latency within F4/80+ cells. Thus, LXRα restricts gammaherpesvirus reactivation through a novel mechanism that is independent of the known CD8+ T cell-based antiviral responses or changes in lipid synthesis and likely involves changes in the tropism of MHV68 in the peritoneal cavity.IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand-activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here, we demonstrate that LXRα, one of the two LXR isoforms, restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

Prognosis in primary effusion lymphoma is associated with the number of body cavities involved.

Primary effusion lymphoma (PEL) is a rare lymphoma associated with Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), and characterized by a malignant body cavity effusion without solid organ or nodal involvement. Prognostic factors in patients with PEL have not been systematically studied. We conducted a literature search for patients with HHV8-positive PEL to identify potential prognostic factors for survival. Our search identified 147 patients, among which 104 patients were HHV8-positive. The median overall survival was 9 months. The median age was 57 years with a male predominance (6:1). Pathologically, 33% of the patients expressed CD20 and 69% expressed CD30. Patients with PEL with > 1 body cavity involved had a median overall survival (OS) of 4 months compared with 18 months in patients with only one cavity involved (p = 0.003). Additionally, in patients with one involved body cavity, pericardial involvement was associated with a longer median OS than pleural followed by peritoneal involvement (40, 27 and 5 months, respectively; p = 0.04). In conclusion, our study suggests that the number and location of body cavities involved are prognostic in patients with PEL.

Antibody-independent control of gamma-herpesvirus latency via B cell induction of anti-viral T cell responses.

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine gamma-herpesvirus 68 (gammaHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell-/- mice during the early phase of gammaHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell-/- mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of gamma-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection.

Biodistribution of oncolytic measles virus after intraperitoneal administration into Ifnar-CD46Ge transgenic mice.

In support of a proposed phase I clinical trial, we studied the biodistribution of virus-infected cells after intraperitoneal administration of oncolytic measles viruses to alpha/beta interferon-defective mice expressing human CD46 with human-like tissue specificity. Various marker genes were employed, and green fluorescent protein proved to be most informative. Mesothelium and ovarian surface epithelium were remarkably resistant to infection, but infected peritoneal macrophages were present in abundance both in peritoneal lavage fluid and in the greater omentum, where they were heavily concentrated in "milky spots". Infected macrophages were also identified outside the peritoneal cavity, along the peritoneal fluid drainage pathway and in the spleen. Thus, diaphragmatic stomata, thoracic lymphatic vessels, and parathymic lymph nodes contained numerous measles-infected cells, as did the marginal zones of the white pulp of the spleen. Splenic marginal zone macrophages were the predominant targets of infection after intravenous administration of oncolytic measles viruses. When measles-infected peritoneal macrophages were adoptively transferred, they did not migrate beyond the confines of the peritoneal cavity, suggesting that, after intraperitoneal virus administration, the positive cells in thoracic lymphatics, parathymic lymph nodes, and spleen are nonmigratory cells transduced in situ by viral particles that have exited from the peritoneal cavity.

Characterization of a spontaneous 9.5-kilobase-deletion mutant of murine gammaherpesvirus 68 reveals tissue-specific genetic requirements for latency.

Murine gammaherpesvirus 68 (gammaHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While gammaHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of gammaHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available gammaHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

Epstein-Barr virus DNA quantitation assessed by a real-time polymerase chain reaction in a case of Burkitt's lymphoma.

A real-time PCR technique was used to quantify EBV DNA load in plasma, leukocytes, peritoneal cells, ascites and cerebrospinal fluid (CSF) at diagnosis and during the follow-up of a 21-year-old patient suffering from an abdominal form of EBV-associated Burkitt's lymphoma. The EBV DNA load correlated well with the clinical and biological remission status of the patient after chemotherapy confirming that EBV DNA quantitation in plasma and leukocytes from peripheral blood can be considered as a marker of the tumor load and can be analyzed in parallel for monitoring of EBV-related malignancies.

The role of LY49 NK cell subsets in the regulation of murine cytomegalovirus infections.

The distributions and functions of NK cell subsets, as defined by the expression of Ly49 NK cell receptors, were examined in murine CMV (MCMV)-infected mice. MCMV induced a reduction in NK1.1+ cell number in the spleen and an increase in the peritoneal exudate cells. Within the splenic NK1.1+ population, proportional increases in Ly49A+ and Ly49G2+ cells but decreases in Ly49C+ and Ly49D+ cells were observed 3 days post-MCMV infection, but within the peritoneal NK1.1+ cell populations there were proportional decreases in Ly49A+ cells and increases in Ly49C+, Ly49D+, and Ly49G2+ cells. Lymphocytic choriomeningitis virus did not elicit a comparable NK cell subset distribution. Lymphokine-activated killer cells were sorted into different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice. Regulation of MCMV synthesis in these suckling mice was shown to be an IFN-gamma-dependent, perforin- and Cmv-1-independent process, and each NK cell subset mediated anti-viral activity. In adult C57BL/6 mice, the control of MCMV in the spleen is mediated by a perforin-dependent mechanism, regulated in part by the Cmv-1 gene, which maps closely to the Ly49 family. In vivo depletions of either one or two of the Ly49 subsets in adult mice did not affect the ability of the residual NK cells to regulate MCMV synthesis. These data provide evidence of NK cell subset distribution and function in MCMV infection, but no individual subset was required for the Cmv-1-like regulation of MCMV synthesis.

Suppression of HIV-1 infection in linomide-treated SCID-hu-PBL mice.

BACKGROUND: Proinflammatory cytokine overproduction, as well as synthesis of the inducible form of nitric oxide synthase (iNOS), are known to play a major role in HIV-1-triggered disease. AIDS patients show increased serum tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels, which synergize with HIV-1-produced nitric oxide (NO) to augment viral replication. Linomide has strong immunomodulatory effects in animals and humans, yielding promising clinical benefits in several pathological disorders including septic shock and autoimmune disease, processes largely mediated by overproduction of these cytokines. In peripheral T cells, linomide also prevents apoptosis triggered by a variety of stimuli, including superantigens, dexamethasone and vaccinia virus. DESIGN AND METHODS: Linomide inhibits production of proinflammatory cytokines such as TNF-alpha, interleukin-1 beta and IFN-gamma, as well as iNOS synthesis. The SCID-hu-PBL mouse model was used to analyse the effect of linomide on HIV-1 infection. T-cell frequency was characterized in reconstituted animals, and the frequency of infected mice and viral load of infected animals were studied. RESULTS: Linomide promotes an increase in human CD4+ T-cell counts in the peritoneal cavity of HIV-1-infected, linomide-treated mice. Linomide also prevents human TNF-alpha and IFN-gamma production, as well as iNOS expression and affects the viral load, promoting potent suppression of HIV-1 infectivity as detected in peritoneal cavity and spleen. CONCLUSIONS: The combination of linomide's properties, namely, blockage of proinflammatory cytokine and NO production, as well as prevention of apoptosis, is of paramount interest, making linomide a potential candidate for combating HIV-1 infection or preventing some of its associated pathological manifestations.

Severe combined immunodeficient mice engrafted with macaque peripheral blood leukocytes support replication of SIVsmm.

Peripheral blood leukocytes (PBLs) from normal pigtail macaques were engrafted into severe combined immunodeficient C.B-17 scid/scid (SCID) mice to develop a small animal model in which to study and identify genetic determinants responsible for the acutely lethal disease syndrome induced by SIVsmmPBj14 (SIV-PBj14) in pigtail macaques. In vivo infection of macaques with SIV-PBj14 results in acute disease in all animals and death of most animals, depending on the route of infection, due to immune activation and production of inflammatory cytokines. A small animal model in which a similar acute disease syndrome was induced would facilitate screening of virus variants to identify regions of the SIV-PBj14 genome responsible for the unique phenotype. Although intraperitoneal inoculation of SCID mice with SIV-PBj14-infected PBLs or uninfected PBLs followed by cell-free SIV-PBj14 produced chimeric mac-PBL-SCID mice that supported SIV replication, obvious clinical signs of disease were not observed. SIV-infected macaque PBLs were recovered from spleen, bone marrow, peripheral blood, and the peritoneal cavity; cell-free SIV was recovered from peritoneal lavage fluid and serum or plasma. PBLs that were mitogen stimulated and SIV-PBj14 infected in vitro migrated rapidly and were recovered from the spleen and bone marrow as early as 1 day after inoculation of mice. The mac-PBL-SCID model may be useful for screening potential drug or immunomodulatory therapies before testing in macaques.

Distribution of mouse adenovirus type 1 in intraperitoneally and intranasally infected adult outbred mice.

In situ nucleic acid hybridization and immunohistochemistry were used to determine the histological localization of mouse adenovirus type 1 (MAV-1) during acute infection of adult mice infected either intraperitoneally or intranasally with 1,000 PFU of wild-type virus. Organ samples were collected from days 1 to 17 postinfection for the intraperitoneally infected mice and from days 1 to 13 for the intranasally infected mice. Endothelial cells of the brain and spinal cord showed extensive evidence of MAV-1 infection. Endothelial cells in lungs, kidneys, and other organs were also positive for MAV-1, indicating a widespread involvement of the systemic circulation. The presence of viral nucleic acid and/or antigen was also demonstrated in lymphoid tissue. The spleens, Peyer's patches, and peripheral lymph nodes showed positive staining at various times postinfection in mice infected by either route. Virus-infected cells in the spleen exhibited a stellate shape and were localized to the red pulp and germinal centers, suggesting that they are cells of the mononuclear phagocytic system.

Viral susceptibility of a newly established cell line derived from the peritoneal cavity of BALB/C mouse.

Viral susceptibility of a newly established cell line, named KMP, derived from the peritoneal cavity of BALB/C mouse is described. The cells were originally cloned from the in vitro culture of ascites of the mouse injected with Ehrlich ascitic tumor cells in advance. The electrophoretic pattern of cellular DNAs, extracted from KMP, normal BALB/C mouse spleen, and Ehrlich tumor cells respectively were compared after triple digestions with restriction endonucleases. This cell line was proved to be of mouse origin, but not the sub-line of Ehrlich tumor cells. The strains of Coxsackie virus B group, swine enterovirus, influenza virus, encephalomyocarditis virus, vesicular stomatitis virus, and Aujeszky's disease virus were able to multiply well in the cell line with considerably high infectious titers in showing clear CPE and circular plaques.

N-acetyl-cysteine and L-2-oxothiazolidine-4-carboxylic acid enhance contact-dependent growth of HIV in resting peripheral blood mononuclear cells (PBMC) in vitro and increase recovery of HIV from human-PBMC SCID mice.

OBJECTIVES: To ascertain the effects of N-acetyl-cysteine (NAC) and L-2-oxothiazolidine-4-carboxylic acid (OTC) on HIV replication in resting T lymphocytes mixed with chronically infected U1 promonocytic cells; examine the phenotypes of NAC- and OTC-treated cells; and monitor HIV recovery from hu-PBMC SCID mice (SCID mice infected with HIV-1BaL reconstituted with human peripheral blood mononuclear cells) treated with oral OTC. DESIGN AND METHODS: Unstimulated PBMC from uninfected donors preincubated for 2 days with pH-adjusted NAC or OTC were cultured at a concentration of 1 x 10(6) cells/ml with 100 U1 cells that were chronically infected with HIV-1IIIB. HI-1 production in the presence or absence of zidovudine was measured by p24 assay at 1-3 weeks, and results were compared with values from the same cell cultures maintained without NAC or OTC exposure. In some experiments U1 cells were separated from PBMC by a 0.4 micron membrane. NAC-treated and -untreated cells were subjected to FACS analysis of multiple-cell-surface adhesion and activation molecules and the results were compared. Hu-PBMC SCID mice were fed OTC for 3 days prior to infection with HIV-1BaL and for the next 3 weeks. Mice were then sacrificed and peritoneal lavage cells were cultured for virus analysis. RESULTS: Unstimulated, non-dividing PBMC supported high levels of HIV replication when in direct contact with U1 cells in the presence of NAC or OTC; CD2 and CD54 (I-CAM1) were down-regulated on NAC-treated PBMC; and OTC-treated mice produced significantly higher yields of HIV-1 from peritoneal cells than did untreated mice. CONCLUSIONS: At concentrations < or = 5 mM, NAC and OTC potentiate HIV growth in unstimulated PBMC in vitro and in SCID mice. Caution in the use of these agents as antiviral monotherapies is advisable.

Highly virulent strains of herpes simplex virus fail to kill mice following infection via gingival route.

Virulence of herpes simplex virus (HSV) in mice has been demonstrated to be dependent on the site of infection. In this experiment, pathogenesis of HSV was studied in 2 different routes of infection in a mouse model system. When BALB/c mice were infected with 5 x 10(3) plaque-forming units (PFU) of virulent HSV type 1 Miyama GC+ strain (HSV-1-GC+) intraperitoneally, all mice were dead in 6 to 9 days. HSV-1-GC+ was recovered from organs such as the cerebrum, cerebellum, brainstem, and spleen 2 to 5 days after infection, but not from other organs such as trigeminal ganglia. However, if mice were infected in the maxillary gingiva with 1.0 x 10(7) PFU of HSV-1-GC+, all mice survived. HSV-1-GC+ was recovered from the trigeminal ganglia and brainstem 2 to 5 days after infection, but not from other organs tested. When mice were infected in maxillary gingiva with HSV-1-GC+, followed by the intraperitoneal injection of 6 mg of cyclophosphamide 72 hrs after virus infection, all mice were dead within days. Immunofluorescent and hematoxylin-eosin staining of gingival tissue sections revealed that when mice were infected in maxillary gingiva with HSV-1-GC+, 3 times as many gamma delta T-cells and 5 times as many polymorphonuclear cells can be detected in sections of maxillary gingiva when compared with non-infected mice. These data show that the gingiva of mice is considerably more resistant to infection with HSV, compared with the peritoneal cavity, and suggest the possible presence of an oral defense mechanism which might be different from that in the peritoneal cavity.

The UL55 and UL56 genes of herpes simplex virus type 1 are not required for viral replication, intraperitoneal virulence, or establishment of latency in mice.

A critical determinant for intraperitoneal virulence of Herpes Simplex Virus Type 1 (HSV-1) strain F has been ascribed to the BamHI-B fragment of the genome. This region contains two nonessential genes, UL55 and UL56, of unknown function. To investigate the contribution of these genes to viral pathogenesis, we have generated two recombinant HSV-1 viruses, inUL55 and inUL56, in which the corresponding gene was inactivated in strain F by insertion of an ICP6::lacZ cassette. Strains inUL55 and inUL56, which did not express the UL55 and UL56 RNA, respectively, were virulent in mice inoculated intraperitoneally. Strains inUL55 and inUL56 also established latent infections in the trigeminal ganglia of mice inoculated via the cornea. These results demonstrate that the HSV-1 UL55 and UL56 genes are not critical for intraperitoneal virulence or establishment of latent infection.

Role of macrophages, interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection.

Genetic heterogeneous mouse populations selected for high (HIII) and low (LIII) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The HIII mice, when compared to their LIII mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of HIII mouse macrophages infected with MHV3. The activation of HIII mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of LIII mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in LIII macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.