Enhancement of viral pathogenesis in mice maintained in an antiorthostatic suspension model: coordination with effects on interferon production

Both rodents and men returning from spaceflight and the attendent microgravity or weightlessness conditions have exhibited alterations in immune responses and, in particular, interferon production. We have utilized a model for antiorthostatic (20 degrees head-down tilt). hypokinetic, hypodynamic suspension of mice that simulates some aspects of weightlessness. Female Swiss/Webster mice that are normally resistant to infection with the D variant of encephalomyocarditis virus showed a marked increase in susceptibility to infection when suspended. This correlated with a drop in interferon production. Control, orthostatically suspended mice (no head-down tilt) showed no increase in susceptibility to the virus. These data suggest that maintenance of mice in the antiorthostatic suspension model for simulating some aspects of weightlessness yielded increased susceptibility to virus infection that was coincident with inhibited interferon production.     

Adaptive Responses of Secretory Cardiomyocytes of the Right Atrium during Simulation of Microgravity Effects by Long-Term and Repeated Antiorthostatic Tail Suspension of Rats

Reactive changes in right atrial cardiomyocytes during antiorthostatic tail suspension of rats, which is commonly used to simulate effects of microgravity, have been studied by electron microscopy and morphometry. A 14-day tail suspension proved to increase contractile and secretory activities of cardiomyocytes. At the same time, signs of depleted activity are observed in some cells. Prolongation of the experiment to 30 days leads to development of adaptive compensatory responses and increases their secretory capacity. A 30-day rest in the normal orthostatic position does not completely restores the structure and functioning of cardiomyocytes and leads to accumulation of internal secretion in them. A repeated 14-day tail suspension to a certain extent facilitates cardiomyocyte adaptation to altered conditions as compared to a single exposure; apparently, secretion release decreases, while its production is activated.     

Suspension osteopenia in mice: Whole body electromagnetic field effects

Whole-body fields were tested for their efficacy in preventing the osteopenia caused by tail suspension in mice. The fields had fundamental frequencies corresponding to the upper range of predicted endogenous impact-generated frequencies (0.25–2.0 kHz) in the long bones. Three distinct whole-body EMFs were applied for 2 weeks on growing mice. Structural, geometric, and material properties of the femora, tibiae, and humeri of suspended mice were altered compared to controls. Comparison of suspended mice and mice subjected to caloric restriction indicates that the changes in caloric intake do not explain either the suspension or the field-induced effects. In agreement with past studies, rather, unloading appears to cause the suspension effects and to be addressed by the EMFs. The EMF effects on bone properties were apparently frequency dependent, with the lower two fundamental frequencies (260 and 910 Hz) altering, albeit slightly, the suspension-induced bone effects. The fields are not apparently optimized for frequency, etc., with respect to therapeutic potential; however, suspension provides a model system for further study of the in vivo effects of EMFs. © 1995 Wiley-Liss, Inc.     

A mouse model of the pathogenesis and postexposure prophylaxis of rabies

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.     

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.     

Functional hypersplenism in mice induced by adoptive transfer of syngeneic spleen cells

Investigation of peripheral blood cell count alterations in cases with hypersplenism, and an understanding of the relationship between splenic function and hematopoietic cell production require suitable experimental animal models. Previously described methods are either traumatic or require surgical intervention. We suggest a relatively simple method for achievement of a state mimicking hypersplenism in mice by intraperitoneal inoculation of syngeneic spleen cells. Mice were inoculated intraperitoneally with 3 x 10(7) splenocytes suspended in 0.3 ml phosphate buffered saline (PBS). After 2 months, the inoculated animals showed a progressive decrease in the peripheral white blood cell (WBC) counts and hyperplastic bone marrow that persisted until the experimental end point (7 months). Five days after inoculation of splenocytes stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE), the majority of the stained cells was present in the peritoneal cavity (33%) and in the liver (13%), whereas the percentage of stained cells in the peripheral blood and the spleen cell suspension was negligible. The mitogen response of the peripheral blood mononuclear cells (PBMC) from treated mice to concanavalin A (Con A) remained unaltered. Splenocyte-inoculated mice that were further splenectomized did not show leukocytosis after splenectomy, as was observed in animals in which the spleen was removed without any pretreatment. The lack of any signs of discomfort in animals from the study group, in comparison with the visibly ill appearance and even death of mice in which hypersplenism was achieved by repeated injections of methylcellulose (MC), which served as controls, favors the convenience of the method.     

Specific binding of atrial natriuretic factor to renal glomeruli during day or night antiorthostatic suspension in the rat

We observed a significant increase in plasma atrial natriuretic factor (ANF) in antiorthostatic hypokinetic suspension (AOH) rats after 2 h of suspension when the experiment was made during day. Plasma ANF was investigated in relation to renal glomerular ANF receptors during AOH at night. The aim of this study was 1) to compare the day and night ANF responses to AOH 2) to determine whether the renal glomerular ANF receptors are involved. The rats were divided into 2 groups: i) 24 population cage (PC), and ii) 24 were attached by the tail (Morey's model) and remained in the horizontal position (attached horizontal-AH). Six AH were suspended (30 degrees) for 2 hours (AOH) and sacrificed with the controls: PC and AH (12.00h). The same experiment was made during the night (24.00h). A significant increase in plasma ANF was found in both AOH and AH after 2 h of suspension during day and night (19 +/- 2.3 pg/ml vs 9 +/- 0.95 and 18 +/- 3 pg/ml vs 10.2 +/- 1.8 respectively). PC rats had a significantly higher ANF level (38 +/- 5 pg/ml) than AH or AOH. The glomerular ANF receptor population was slightly lower in AOH than in AH (429 +/- 12 fmol/mg protein vs 507 +/- 5) during day. During night, a significantly lower number of ANF receptors was observed in AOH animals as compared to AH (168 +/- 2 fmol/mg protein vs 455 +/- 3). A decrease in glomerular receptors was also noted in PC during night. Day-time head-down tilt, bed rest or head-out water induced a natriuretic and diuretic response, whereas the normal recumbency at night does not lead to such effects. We conclude that the natriuretic and diuretic response not observed during night was associated with elevated plasma ANF levels and decreased ANF receptor density.     

The realization of a mechanical signal during gravitational unloading: The response of mTORC1 targets to eccentric contractions

The aim of this study was to assess the response of key mTORC1 substrates to a bout of contractile stimuli under different times of functional unloading. Functional unloading of hind-limb muscles was carried out by the method of antiorthostatic suspension. Twenty-eight Wistar rats were divided into four groups: control, and hindlimb suspension for 1, 3, and 7 days. After hindlimb suspension, isolated soleus muscles of rats were subjected to a bout of ex vivo eccentric contractions. The contents of phosphorylated forms of p70s6k and 4E-BP1 were then determined using western blotting. It was found that an eccentric load resulted in a significant increase in p70s6k phosphorylation and reduced 4E-BP1 phosphorylation both in control and suspended rats, but in the case of suspension the response was dramatically reduced. Thus, it can be concluded that a bout of eccentric contractions of isolated rat soleus muscle during functional unloading causes a weaker activation of the Akt-mTORC1-p70s6k signaling pathway compared with the control. This may indicate that it is important to maintain muscle tone for a more efficient muscle perception of an external mechanical signal and subsequent activation of anabolic signaling pathways.     

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.     

Protection by dexamethasone from a lethal infection with Listeria monocytogenes in mice

Abstract The effects of dexamethasone (DEX) on a lethal infection with Listeria monocytogenes were studied in mice. Mice were completely protected against the lethal infection when treated with 3.3 mg per kg of DEX. The effect was observed only when DEX was injected before infection. The control mice died from day 3 to day 5 of infection, whereas DEX-treated mice could eliminate L. monocytogenes cells from the organs by day 11 of infection. High titres of endogenous tumour necrosis factor (TNF), interleukin-6 (IL-6) and gamma interferon (IFN-γ) were induced in the bloodstreams and organs of the drug-free mice. DEX suppressed IL-6 production, but augmented TNF and IFN-γ production within 24 h of infection, whereas production of all three endogenous cytokines was suppressed in the DEX-treated mice on day 3 of infection when the control mice began to die. These results suggest that DEX shows a protective effect on a lethal infection with L. monocytogenes in mice and that regulation of production of endogenous cytokines might be involved in the effect of DEX.     

Accumulation of 2'',5''-oligoadenylates in encephalomyocarditis virus-infected mice.

Levels of 2',5'-oligoadenylates (2-5A) in various tissues of murine encephalomyocarditis virus (EMCV)-infected mice were determined and compared with those found in pathogen-free mice and in mice treated with the interferon inducer poly(I).poly(C). In control, pathogen-free mice, liver, spleen, brain, and kidney tissues possessed levels of 2-5A below 1 pmol/g of tissue, demonstrating that 2-5A was not a major component of uninfected mouse tissue. All control tissues had low basal levels (0.3 to 2.0 pmol/h per g) of 2-5A synthetase, the enzyme responsible for 2-5A production. After mice were injected intravenously with the interferon inducer poly(I).poly(C), circulating interferon, 2-5A synthetase, and 2-5A were elevated with increasing doses of double-stranded RNA. The greatest response to poly(I).poly(C) occurred in the kidney, in which enzyme levels increased 5-fold and 2-5A levels increased 24-fold to 15 pmol/g. Mice that were infected with EMCV also possessed elevated levels of 2-5A and 2-5A synthetase in the four tissues examined, although the relative distribution differed from that observed with poly(I).poly(C), indicating that the interferon inducer affects the concentration and location of intracellular 2-5A. Brain, spleen, and kidney tissues from EMCV-infected mice contained seven- to eightfold more 2-5A than control tissues did. The nanomolar levels of 2-5A in the tissues of EMCV-infected mice provide evidence that 2-5A may play a role in the antiviral response in an intact animal. In both poly(I).poly(C)- and EMCV-treated mice, the levels of 2-5A recovered from the tissues were not directly proportional to the amount of 2-5A synthetase present. These results indicate that factors other than the level of 2-5A synthetase controlled the accumulation of 2-5A in tissues.     

Signaling through the prostaglandin I2 receptor IP protects against respiratory syncytial virus-induced illness

The role of prostanoids in modulating respiratory syncytial virus (RSV) infection is unknown. We found that RSV infection in mice increases production of prostaglandin I(2) (PGI(2)). Mice that overexpress PGI(2) synthase selectively in bronchial epithelium are protected against RSV-induced weight loss and have decreased peak viral replication and gamma interferon levels in the lung compared to nontransgenic littermates. In contrast, mice deficient in the PGI(2) receptor IP have exacerbated RSV-induced weight loss with delayed viral clearance and increased levels of gamma interferon in the lung compared to wild-type mice. These results suggest that signaling through IP has antiviral effects while protecting against RSV-induced illness and that PGI(2) is a potential therapeutic target in the treatment of RSV.     

Influenza virus-induced type I interferon leads to polyclonal B-cell activation but does not break down B-cell tolerance

The link between infection and autoimmunity is not yet well understood. This study was designed to evaluate if an acute viral infection known to induce type I interferon production, like influenza, can by itself be responsible for the breakdown of immune tolerance and for autoimmunity. We first tested the effects of influenza virus on B cells in vitro. We then infected different transgenic mice expressing human rheumatoid factors (RF) in the absence or in the constitutive presence of the autoantigen (human immunoglobulin G [IgG]) and young lupus-prone mice [(NZB x NZW)F(1)] with influenza virus and looked for B-cell activation. In vitro, the virus induces B-cell activation through type I interferon production by non-B cells but does not directly stimulate purified B cells. In vivo, both RF and non-RF B cells were activated in an autoantigen-independent manner. This activation was abortive since IgM and IgM-RF production levels were not increased in infected mice compared to uninfected controls, whether or not anti-influenza virus human IgG was detected and even after viral rechallenge. As in RF transgenic mice, acute viral infection of (NZB x NZW)F(1) mice induced only an abortive activation of B cells and no increase in autoantibody production compared to uninfected animals. Taken together, these experiments show that virus-induced acute type I interferon production is not able by itself to break down B-cell tolerance in both normal and autoimmune genetic backgrounds.     

Influence of hindlimb suspension on calcium-induced contraction characteristics in dystrophin-deficient animals

The direct data concerning effects of unloading on dystrophic muscle were received in study of mdx mice, a model for Duchenne muscular dystrophy, muscles before and after hindlimb suspension. Experiments were performed on softer skinned soleus muscle fibers isolated from wild-type (C57black) as a control and mdx mice aged 2 weeks. Animals of two experimental groups were tail suspended during 21 days. In both groups of hindlimb suspended mice isolated soleus fibers were thinner than in the control groups. But there was a greater 37% significant decrease in fiber diameter in wild-type (CHS) suspended mice vs. 24% in mdx (MHS) suspended group. Values of absolute peak tension in CHS were less than in the control group by 33%, and in MHS mice suspended--by 39%. 21 days of hindlimb suspension resulted in reduction of mean peak specific tension by 28% in MHS and significantly less drop (15%) in CHS groups. We observed a similar rightward shift of the tension pCa curve in both mice strains.     

Zoledronic acid repolarizes tumour‐associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB‐2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour‐free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour‐associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin‐10 was drastically down‐regulated in favour of interferon‐γ production. Peritoneal macrophages and tumour‐associated macrophages of ZA‐treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour‐associated macrophages and their reverted polarization from M2 to M1 phenotype.     

Pulmonary compartmentalization of interferon and natural killer cell activity

Studies were performed to determine if natural killer (NK) activity in the mononuclear cells harvested from infected lungs was dependent on local or systemic factors. Mice were inoculated by intratracheal (it), intraperitoneal (ip), or intravenous (iv) routes with (a LD50 dose of) influenza virus A PR/8/34. At various days postinoculation cells from lungs, spleens, and peripheral blood were assayed for NK activity, and lung wash, lung homogenates, and serum were assayed for interferon. After it inoculation there was three- to fourfold increase of NK activity in the lung with little or no increase in NK activity in spleens or peripheral blood. The local augmentation of NK activity in the lung correlated with an increase in interferon (IFN) titer in the lung wash and lung homogenate of PR8 inoculated mice. The virus failed to induce IFN or augment NK activity when it was inoculated systemically. The observed local augmentation of NK activity and local induction of interferon production following it inoculation suggests that the NK population in the lung is capable of responding to locally derived regulatory factors.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

The reliable diagnosis for an early stage of hereditary muscular dystrophy of mice (author's transl)

The methods to find hereditary muscular dystrophy mice (dy-mice) were described. One was to observe whether or not mice dragged their hind limbs immediately after they were dropped onto a table following their suspension by their tails. The other was to observe dragging their hind limbs immediately after they were restored to a normal position from a supine one. This limbs-dragging induced by these methods were characteristic of dy-mice. Among 437 mice checked by these methods, 76 showed the drag during 14-23 days after birth (18.3 days on the average) and later they were confirmed to be dy-mice without exception. Therefore, it was concluded that these methods were useful for the accurate diagnosis of dy-mice at an early stage.     

A monoclonal antibody against T-cell receptor αβ induces endogenous cytokines and prevents mice from a lethal infection with Listeria monocytogenes

Abstract In vivo induction of cytokines by a monoclonal antibody (mAb) against T-cell receptor (TCR) αβ and the protective effect induced by the mAb on a lethal infection with Listeria monocytogenes were studied. Injection of anti-TCR αβ mAb induced rapid production of endogenous tumour necrosis factor in the spleens, and gamma interferon and interleukin-6 in the bloodstreams and spleens of mice. Administration of anti-CD4 mAb, anti-CD8 mAb, or anti-Thy1.2 mAb resulted in suppression of anti-TCR αβ mAb-induced endogenous cytokine production. Mice were protected against lethal L. monocytogenes infection when treated with anti-TCR αβ mAb. The protective effect was not demonstrated in CD4 + cell- or CD8 + cell-depleted mice. These results suggest that anti-TCR αβ mAb shows a protective effect on a lethal infection with L. monocytogenes in mice and that the mAb-induced endogenous cytokines might be involved in the effect of anti-TCR αβ mAb.