Mitochondria-related gene expression profiles in murine fibroblasts and macrophages during later stages of ectromelia virus infection in vitro.

Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of ectromelia virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: ectromelia virus; mitochondria; fibroblasts; macrophages.

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

In vivo induction of autophagy in splenocytes of C57BL/6 and BALB/c mice infected with ectromelia orthopoxvirus.

Autophagy is a self-degradation process of cellular components. It plays both antiviral and pro-viral roles in the life cycle of different viruses and the pathogenesis of different viral diseases. In this study, we evaluated autophagy induction in splenocytes of ectromelia virus (ECTV)-resistant C57BL/6 and ECTV-susceptible BALB/c mice during infection with the Moscow strain of the ectromelia virus (ECTV-MOS). Autophagy was analyzed using the Western blot method by assessing type II microtubule-associated protein 1 (MAP1) light chain 3 (LC3) and Beclin 1 expression levels relative to beta-actin. Results indicated an increased ratio of LC3-II to beta-actin in splenocytes of C57BL/6 mice only at 7 day post infection (d.p.i.) compared to uninfected animals. LC3-II/beta-actin and Beclin 1/beta-actin ratios in splenocytes of BALB/c mice increased at 5 d.p.i. and remained high until day 14 and 7 p.i., respectively. We confirmed the formation of autophagosome structures in the spleen of BALB/c mice by transmission electron microscopy (TEM). Moreover, autophagy accompanied necrosis in the splenocytes of infected animals. Results suggest that ECTV-MOS induced autophagy, especially in the spleen of the susceptible mouse strain, may support viral replication and promote cell necrosis.

The effect of multigenerational diet containing genetically modified triticale on immune system in mice.

The safety assessment of genetically modified (GM) food and feed is performed to identify the possible effects upon animal and human health, also the long-term, multigenerational influence upon functioning of different organs and systems, such as the immune system. In this study C57BL/6J mice were fed for five consecutive generations with pellets containing 20% of conventional triticale grain (control) vs. pellets containing 20% of the transgenic triticale grain resistant to BASTA herbicide (experimental). The F5 experimental animals showed enlarged inguinal and axillary lymph nodes, but not spleens, and increased WBC counts in blood (but within the norm for Mus musculus). Immunophenotyped cell suspensions derived from spleens, inguinal and axillaris lymph nodes and PBMCs from blood showed the significant decrease in the percentage of T cells in spleen and lymph nodes and the B cells in lymph nodes and blood of the F5 experimental mice in comparison to the control F5 mice. Immunoblotting analysis of IL-2, IL-4, IL-10, IL-12, IL- 6, IFN-gamma levels in serum showed significantly increased IL-2 levels and decreased IL-6 levels in the F5-experimental mice sera. No significant changes in the levels of IgE in sera in both mice groups were observed. The obtained results indicate that multigenerational use of feeds for rodents containing the GM-triticale leads to expansion of the B cell compartment in the secondary lymphoid organs, but it is not caused by malignant processes or the allergic response.

Antigen presenting and effector cell cluster formation in BALB/c mice during mousepox: model studies*.

AIMS: The objective of this study was to access APC-effector cell cluster formation in genetically susceptible BALB/c (H-2(d) ) mice infected with highly virulent Moscow strain of ectromelia virus (ECTV-MOS) and estimate of lymphocyte activation based upon expression of CD62L and CD44 molecules. METHODS AND RESULTS: APC-effector cell clusters were obtained by enzymatic digestion from draining lymph nodes (DLNs) and spleens of BALB/c mice. We found that APCs infected with ECTV-MOS form unstable clusters with effector cells, and thus may diminish T-cell activation at the early stage of mousepox. Different types of effector cells including T-cell subsets (CD4(+) and CD8(+) ), B cells and polymorphonuclear cells colocalize within individual clusters. Increase in CD19(+) B cells within APC-effector cell clusters during severe clinical mousepox may reflect B-cell activation. CONCLUSIONS: Our studies indicated vigorous changes in APC-effector cell cluster formation in genetically susceptible BALB/c mice during mousepox (up to 2 weeks). ECTV-MOS can modulate APC interactions with effector cells and consequently may impair T-cell activation probably owing to unstable cluster formation and/or subsequent weak stimulation by infected APCs at the early stages of mousepox. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of APC-effector cell cluster formation in BALB/c mice during mousepox. It gives us a new light on the mutual cell-cell interactions and development of the immune response during ECTV-MOS infection.

Contribution of rearranged actin structures to the spread of Ectromelia virus infection in vitro.

We describe here a contribution of virus-induced actin tails and filopodia in transmission of Ectromelia virus (ECTV) infection in permissive cells detected by the immunofluorescence and confocal microscopy. Immunoblot analysis revealed profoundly decreased beta-actin levels during ECTV replicative cycle in the infected cells 24 hrs post infection (p.i.). These results provided a basis for the further analysis of ECTV motion in the infected cells as well as for impact of ECTV infection on the cytoskeletal proteins.

Immunobiology of bovine mammary gland: apoptosis of somatic cells in milk during naturally occurring mastitis.

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.

Pathogenesis of mousepox in H-2(d) mice: evidence for MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) CTL antiviral activity in the lymph nodes, spleen and skin, but not in the conjunctivae.

Genetically sensitive mice (i.e. H-2(d) haplotype) infected with a natural mouse pathogen named ectromelia virus (EV) can develop a mousepox. Virus replicates well in the skin, next in the draining lymph nodes (DLNs) and then in the spleen and liver, where it may induce extensive necrosis with strong inflammatory reaction. It is well known from the studies defined on some other viruses that a correlation, functional link and powerful help exist between MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) virus-specific cytotoxic T lymphocytes (CTLs). However, in the case of mousepox the role of CD4(+) CTLs is still controversial and some reports support the notion that induction of EV-specific CD4(+) CTLs is nonessential for the generation of virus-specific immune response. Consequently, this study was designed to evaluate EV-specific CD8(+) and CD4(+) CTL activity in the DLNs, spleen, skin and conjunctivae of BALB/c (H-2(d)) mice at 7 and 14 days p.i. with Moscow strain of EV. By using bulk cytotoxicity assay and immunosurgery of effector T cells with mAb specific for CD4(+) and/or CD8(+) T cells our data show that EV-specific CD8(+) CTLs predominated in DLNs and spleen at 7 days (67 and 66% of total CTLs, respectively) and 14 days p.i. (63 and 69% of total CTLs, respectively). In contrast, we found that EV clearance from the cutaneous lesions during mousepox is CD4(+) CTL-dependent at 7 days p.i. (59% of total CTLs), whereas at 14 days p.i. CD8(+) CTLs predominated in the epidermis, accounting for 72% of the total EV-specific CTLs. Our studies showed that the population of EV-specific CTLs is heterogeneous and contains cells of both phenotypes: CD8(+) and CD4(+). However, these effector cells did not express a similar tendency in cytotoxic activity in the DLNs, spleen and skin in comparison to the conjunctivae where EV-specific CD8(+) and CD4(+) CTLs were not detected at 7 days p.i. and at peak of mousepox conjunctivitis (14 days p.i.). Our results are discussed in terms of the value of EV to study antiviral CTL responses in the genetically susceptible host.

Homing studies on distribution of ectromelia (mousepox) virus-specific T cells adoptively transferred into syngeneic H-2d mice: paradigm of lymphocyte migration.

Mousepox (infectious ectromelia) may be used as a model for studies on the cellular immune response and pathogenesis of generalized viral infections. Ectromelia virus (EV) initially replicates in the footpad (f.p.) skin at the site of infection, next in draining lymph nodes, and then in the spleen and liver where the virus may induce extensive necrotic process with inflammatory reaction. We show in this study that after recipient BALB/c mice (H-2d) f.p. infection with EV prior to the adoptive transfer of syngeneic donor EV-specific cytotoxic T lymphocytes interferon-gamma-positive (IFN-gamma-+), interleukin-2-positive (IL-2+), and IL-4+ of both phenotypes, CD8+ approximately 70%, and CD4+ approximately 30%) preferentially migrated to the inguinal and auxiliary lymph nodes, spleen, liver, and skin at the site of infection (f.p.). Many particles of EV with the morphology characteristic for orthopoxviruses and virus-specific immunofluorescence within the cells of inguinal and auxiliary lymph nodes, liver, spleen, and skin have been observed using high-resolution transmission electron microscopy and fluorescence antibody technique, respectively. Results presented in this article support the concept that immune T cells adoptively transferred into infected recipient mice are able not only to specific migration in the host and homing in the sites of virus replication, but also to develop immunoprotection in the transferred animals.

How human immunodeficiency viruses and herpesviruses affect apoptosis.

Eighty to hundred percent of patients positive for human immunodefficiency viruses 1 or 2 (HIV) may develop opportunistic viral infections. According to the National Institute of Health data, only in the USA the HIV patients are positive also for human cytomegalovirus (HCMV) in 25-40%, varicella-zoster virus (VZV) in 10%, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8, KSHV, HHV-8) in 20%. HIV and herpesviruses express numerous different proteins that are able to influence interactions between the host and virus. One of the most interesting regulatory phenomenon is apoptosis which could play a significant role during both specific and non-specific antiviral response and latency. Apoptosis is an ordered cascade of precisely regulated enzymatic reactions which may be modulated or even controlled by viruses. Dramatic changes which occur during infection and which are exerted by HIV and certain herpesviruses on the mechanism of apoptosis may contribute to the pathogenesis of acquired immunodeficiency syndrome (AIDS).

Cytotoxic T lymphocyte control during ectromelia (mousepox) virus infection: interaction between MHC-restricted cells analyzed by non-radioactive fluorometry.

Cytotoxic T lymphocyte (CTL) activity of draining lymph node (DLN) cells isolated from BALB/c mice infected with ectromelia virus (EV) was examined using a fluorometric cell-mediated cytotoxicity (CMC) assay. Specific lysis of target cells A20 and EMT-6 primed with EV was demonstrated. The classical CD8+ cytolytic pathway dominated (72.7%) as compared to that of CD4+ (27.3%) in the cellular response during acute EV infection. Also an alternative method for determining CMC, employing a bisbenzamide dye for labelling target cells, is described. Coefficient variations of relative fluorescence were below 6%, that makes the method sensitive and reliable.

The cytosolic free Ca2+ in ectromelia (mousepox) virus stimulated cytotoxic T lymphocytes.

The objective of these studies was to assess the intracellular calcium (Ca2+) signal involved in the activation of ectromelia virus (EV)-specific cytotoxic T lymphocytes (CTL) upon stimulation with EV-sensitized (A20) target cells or concanavalin A (Con A). The CTL originated from mice previously infected with EV. The level of cytosolic Ca2+ in EV-specific CTL was measured using Fluo-3 AM. In both cases transient [Ca2+]i rise and a sustained plateau (1723 nM) were observed in buffer with 1 mM extracellular calcium. The [Ca2+]i response of EV-specific CTL to EV-sensitized target cells or Con A in extracellular calcium free buffer consisted of only one peak at 852 nm. The cytotoxic activity EV-specific CTL assessed in normal medium was 53.0%, but significantly reduced to 15% when verapamil was added to the buffer. No enhancement of [Ca2+]i response in EV-specific CTL was observed upon costimulation with PMA. Association of the level of intracellular [Ca2+]i in EV-specific CTL and the percentage of their cytotoxicity was found. These results show partial dependence of EV-specific CTL killing on extracellular calcium.

Controlling orthopoxvirus infections--200 years after Jenner's revolutionary immunization.

An 11-year global WHO campaign for eradication of smallpox finished in October 1977 as the result of Edward Jenner's primary success in 1796, who for the first time applied human vaccination against variola virus (VARV). The 200th anniversary of this happening is a good occasion to summarize the current status of the knowledge about the role of B and T lymphocytes in the control of orthopoxvirus infections. This short review concentrates on general characteristics of orthopoxviruses and the immune response to infection, mainly by vaccinia virus (VV) and ectromelia virus (EV).

The inflammatory and immune response to mousepox (infectious ectromelia) virus.

The ectromelia virus (EV) has been recognized as the etiological agent of a relatively common infection in laboratory mouse colonies around the world, i.e., Europe (including Poland), USA and Asia. Due to widespread use of mice in biomedical research, it is important to study the biology of strains characteristic for a given country. This is particularly significant for the diagnosis, prevention and control ectromelia. In severe epizootics, approximately 90% morbidity is observed within colonies and mortality rate exceeding 70% is observed within 4 to 20 days from the appearance of clinical symptoms. The resistance to lethal infection is mouse strain-dependent. Several inbred strains of mice, including C57BL/6 and AKR are resistant to the lethal effects of EV infection, while others, such as A and BALB/c are susceptible. Recent studies indicate that (1) T lymphocytes, NK cells and interferon (IFN)-dependent host defenses must operate for the expression of resistance, (2) virus-specific T-cell precursors appear earlier in regional lymph nodes of resistant than susceptible mice, and (3) resistance mechanisms are expressed during early stages of infection. Over the past several years, (1) induction of anti-EV cytotoxic CD8+ T lymphocytes (CTL) responses in vivo in the absence of CD4+ (T helper) cells, (2) importance of some cytokines e.g., IFN-gamma in EV clearance at all stages of infection, and (3) induction of nitric oxide (NO) synthase, which is necessary for a substantial antiviral activity of IFN-gamma, have been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Phagocytic activity of polymorphonuclear leukocytes lavaged from the lungs of horses with clinically diagnosed chronic pulmonary disease.

The aim of this study was to compare phagocytic activity of polymorphonuclear cells (PMNs) from the bronchoalveolar lavage of clinically healthy horses and those with severe chronic bronchiolitis. Research was carried out on 28 horses. Chronic inflammation of the lower airways was diagnosed in nine horses. Cells from the respiratory tract were lavaged according to accepted methods. For comparison, PMNs were isolated from peripheral blood of all investigated horses. The phagocytic activity of PMNs was determined in relation to two standard strains of Staphylococcus aureus, Staph, aureus Smith which was phagocytized after previous opsonization, and Staph, aureus 305, phagocytized without opsonization. From the investigations, it is shown that the PMNs present in the terminal airways of horses with severe chronic bronchiolitis are characterized by decreased phagocytic activity in relation to opsonized Staphylococcus aureus Smith and increased activity in relation to non-opsonized Staphylococcus aureus 305, as compared to the PMNs lavaged from the terminal airways of clinically healthy horses. No changes in the phagocytic activity of the peripheral blood PMNs were observed between clinically diseased horses and healthy horses.

Quantitative studies on CD4+ and CD8+ cytotoxic T lymphocyte responses against herpes simplex virus type 1 in normal and beta 2-m deficient mice.

Against herpes simplex virus type 1 (HSV-1) infected target cells, effector T cells of both CD4- CD8+ and CD4+ CD8- phenotypes occur in both man and mice. In man, the CD4+ CD8- phenotype may dominate the response, but the situation in the mouse is unclear. In this report, several approaches are used to document the existence of HSV-1-specific cytotoxic T lymphocytes (CTL) of both phenotypes and to quantitate the frequency of their CTL precursors (CTL-p) in acutely HSV-1 infected animals. Evidence that CD4+ CD8- CTL occur was confirmed with beta 2-m- mice lacking CD8+ T cells and which generated CTL capable only of lysing MHC class II infected target cells. Comparisons of frequencies of CD4+ CD8- and CD4- CD8+ CTL-p showed the former to be 2-3-fold more frequent. The beta 2-m- mice generated only CD4+ CD8- CTL-p and their frequencies approximately equally the CD4+ CD8- CTL-p frequency in intact infected mice. Our results are discussed in terms of the value of the mouse model to study HSV-1 immunobiology.

Cytotoxic T lymphocyte response to herpes simplex virus type 1 is composed of both CD8+ and CD4+ T cell phenotypes in acute and memory states.

Mice were infected via the cornea with HSV-1. Next, draining lymph nodes (DLN) and spleen cells were analyzed at various times post infection for the presence of cytotoxic T lymphocyte precursors (CTL-p) of both the CD8+ and CD4+ phenotypes. Responses were greatest in the DLN, but memory CTL persisted in the spleen and were undetectable in DLN by 60 days. On all occasions, the frequency of CD8+ CTL outnumbered CD4+ CTL. The murine CTL responses to HSV-1 differ from those in man where CD4+ MHC class II restricted CTL appear to dominate the response at least in the memory phase.