Primary-infected Mice Research Articles

Human Metapneumovirus Reinfection in Aged Mice Recapitulates Increased Disease Severity in Elderly Humans Infected with Human Metapneumovirus.

Human metapneumovirus (HMPV) is a leading cause of respiratory infection in adults >65 y. Nearly all children worldwide are seropositive for HMPV by age 5 y, but reinfections occur throughout life, and there is no licensed vaccine. Recurrent HMPV infection is mild and self-resolving in immunocompetent individuals. However, elderly individuals develop severe respiratory disease on HMPV reinfection that leads to a high risk for morbidity and mortality. In this study, we developed a mouse model to mirror HMPV reinfection in elderly humans. C57BL/6J mice were infected with HMPV at 6-7 wk old, aged in-house, and rechallenged with high-dose virus at 70 wk. Aged rechallenged mice had profound weight loss similar to primary infected mice, increased lung histopathology, and accumulated cytotoxic CD8+CD44+CD62L-CD69+CD103+ memory cells despite having undetectable lung virus titer. When aged mice 14 mo postinfection (p.i.) or young mice 5 wk p.i. were restimulated with HMPV cognate Ag to mimic epitope vaccination, aged mice had an impaired CD8+ memory response. Convalescent serum transfer from young naive or 5 wk p.i. mice into aged mice on day of infection did not protect. Aged mice vaccinated with UV-inactivated HMPV also exhibited diminished protection and poor CD8+ memory response compared with young mice. These results suggest aged individuals with HMPV reinfection have a dysregulated CD8+ memory T cell response that fails to protect and exacerbates disease. Moreover, aged mice exhibited a poor memory response to either epitope peptide or UV-inactivated vaccination, suggesting that aged CD8+ T cell dysfunction presents a barrier to effective vaccination strategies.

Cytomegalovirus reactivation enhances the virulence of Staphylococcus aureus pneumonia in a mouse model

ObjectivesCytomegalovirus (CMV) reactivation in intensive care unit patients may increase mortality and favour bacterial pneumonia. We developed a murine model to compare the severity of staphylococcal pneumonia after CMV reactivation and in CMV-negative mice. MethodsBalb/c mice were primo-infected with murine cytomegalovirus (MCMV n=90) or received saline (control n=90). After latency, all mice underwent caecal ligation and puncture to trigger MCMV reactivation in MCMV primary-infected mice. Surviving animals received an intra-nasal inoculation with methicillin-susceptible Staphylococcus aureus (MSSA) to induce pneumonia. Mortality, lung bacterial count, histology and interferon-alpha and gamma serum levels were compared in MCMV reactivated and control mice 2, 5 and 15 days after pneumonia. ResultsAfter MSSA pneumonia, MCMV mice showed a trend towards a higher mortality (9.4% versus 0%; p 0.09) and a higher weight loss (2.2 (0.6–4.1 g) versus 0.7 (–0.3 to 1.3 g); p 0.005).The lung bacterial count was higher in MCMV mice 2 days (5×103 (103 to 3×105) versus 102 (0 to 4×102) CFU/lung; p 0.007) and 5 days (2.5×104 (1.6×104 to 6.5×105) versus 15 (10–40) CFU/lung; p 0.005) after MSSA pneumonia. 8/40 (20%) MCMV mice developed lung abscesses compared to 0% in control (p 0.011). Interferon-alpha serum levels 2 days after staphylococcal pneumonia were higher in MCMV mice. ConclusionsMCMV reactivation decreased lung bacterial clearance and favoured the development of staphylococcal abscessing pneumonia. CMV reactivation may be responsible for a higher susceptibility to bacterial sepsis.

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.

Vaccination against filarial nematodes with irradiated larvae provides long-term protection against the third larval stage but not against subsequent life cycle stages

Sustainable control of human filariasis would benefit enormously from the development of an effective vaccine. The ability to vaccinate experimental animals, with reductions in worm burden of over 70%, suggests this aim is possible. However, in experimental vaccinations the challenge is usually administered 2 weeks after the immunisation phase and thus the protection obtained is likely to be biased by persisting inflammation. Using the murine model Litomosoides sigmodontis, we increased the time between immunisation with irradiated larvae and challenge with fully infective L3 to 5 months. Significant protection was achieved (54–58%) and the reduced worm burden was observed by 10 days p.i. The developmental stage targeted was the L3, since no nematodes died once they reached the pleural cavity of vaccinated mice, as has been previously shown in short-term protocols. However, larval developmental rate was faster in vaccinated than in primary-infected mice. Immunological assessments were made prior to challenge and then from 6 h to 34 days post-challenge. Samples were taken from the subcutaneous tissue where the larvae were inoculated, the lymph nodes through which they migrate and the pleural cavity in which they establish. Eosinophils were still present although scarce in the subcutaneous tissue of vaccinated mice before challenge. Cytokine and specific antibody production of vaccinated and challenged mice were L3-specific and Th2-biased and greatly exceeded the response of primary-infected mice. The heightened Th2 response may explain the faster development of the filarial worms in vaccinated mice. Thus, long-term vaccination protocols generated a strong memory response that led to significant but incomplete protection that was limited to the infective larval stage suggesting alternative vaccination strategies are needed.

The subcutaneous movements of filarial infective larvae are impaired in vaccinated hosts in comparison to primary infected hosts

Our aim in this study was to observe the movements of filarial infective larvae following inoculation into the mammalian host and to assess the effect of vaccination on larval migration, in situ. Here we present recordings of larvae progressing through the subcutaneous tissues and inguinal lymph node of primary infected or vaccinated mice. We used the filaria Litomosoides sigmodontis in BALB/c mice that were necropsied 6 hours after the challenge inoculation of 200 larvae. Subcutaneous tissue sections were taken from the inoculation site and larvae were filmed in order to quantify their movements. Our analyses showed that the subcutaneous larvae were less motile in the vaccinated mice than in primary-infected mice and had more leucocytes attached to the cuticle. We propose that this reduced motility may result in the failure of a majority of larvae to evade the inflammatory reaction, thereby being a possible mechanism involved in the early vaccine-induced protection.

Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms.

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab')2 fragments.

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.

IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis.

The pathways conferring immunity to human filariases are not well known, in part because human-pathogenic filariae do not complete a full life cycle in laboratory mice. We have used the only fully permissive infection of mice with filariae, i.e., infection of BALB/c mice with the rodent filarial nematode Litomosoides sigmodontis. Our previous results showed that worm development is inversely correlated with Th2 cytokine production and eosinophilia. The scope of the present study was to directly elucidate the role of interleukin-5 (IL-5) and eosinophils in controlling the development of L. sigmodonitis after vaccination and in primary infection. BALB/c mice immunized with irradiated third-stage larvae (L3) were confirmed to have elevated IL-5 levels as well as high subcutaneous eosinophilia and to attack and reduce incoming larvae within the first 2 days, resulting in 70% reduction of worm load. Treatment of vaccinated mice with anti-IL-5 antibody (TRFK-5) suppressed both blood and tissue eosinophilia and completely abolished protection. This demonstrates, for the first time in a fully permissive filarial infection, that IL-5 is essential for protection induced by irradiated L3 larvae. In contrast, in primary-infected mice, anti-IL-5 treatment did not modify filarial infection within the 1st month, most likely because during primary infection IL-5-dependent mechanisms such as subcutaneous eosinophilia are induced too late to disturb worm establishment. However, there is a role for IL-5 late in primary infection where neutrophil-dependent worm encapsulation is also under the control of IL-5.

Effects of reproductive hormones on experimental vaginal candidiasis.

Vulvovaginal candidiasis (VVC) is an opportunistic mucosal infection caused by Candida albicans that affects large numbers of otherwise healthy women of childbearing age. Acute episodes of VVC often occur during pregnancy and during the luteal phase of the menstrual cycle, when levels of progesterone and estrogen are elevated. Although estrogen-dependent experimental rodent models of C. albicans vaginal infection are used for many applications, the role of reproductive hormones and/or their limits in the acquisition of vaginal candidiasis remain unclear. This study examined the effects of estrogen and progesterone on several aspects of an experimental infection together with relative cell-mediated immune responses. Results showed that while decreasing estrogen concentrations eventually influenced infection-induced vaginal titers of C. albicans and rates of infection in inoculated animals, the experimental infection could not be achieved in mice treated with various concentrations of progesterone alone. Furthermore, progesterone had no effect on (i) the induction and persistence of the infection in the presence of estrogen, (ii) delayed-type hypersensitivity in primary-infected mice, or (iii) the partial protection from a secondary vaginal infection under pseudoestrus conditions. Other results with estrogen showed that a persistent infection could be established with a wide range of C. albicans inocula under supraphysiologic and near-physiologic (at estrus) concentrations of estrogen and that vaginal fungus titers or rates of infection were similar if pseudoestrus was initiated several days before or after inoculation. However, the pseudoestrus state had to be maintained for the infection to persist. Finally, estrogen was found to reduce the ability of vaginal epithelial cells to inhibit the growth of C. albicans. These results suggest that estrogen, but not progesterone, is an important factor in hormone-associated susceptibility to C. albicans vaginitis.

Reactive oxygen intermediates from eosinophils in mice infected with Hymenolepis nana.

A large number of eosinophils were recruited to the intestinal villi after infection with Hymenolepis nana. Eosinophil numbers were increased more rapidly in challenged mice than in primary infected mice. Local intestinal eosinophils from challenged mice showed more extracellular oxygen radical release, as assessed by histochemical methods using nitro blue tetrazolium, accompanied with tissue injury and larval degradation. Intestinal eosinophils isolated from the lamina propria induced specific oxygen radical generation in response to H. nana oncosphere extract as measured by luminol-dependent chemiluminescence. This response was stronger in challenged mice than in primary infected mice. Radical generation from uninfected mice was negligible. Lipid peroxidation in the small intestine, as measured by formation of malondialdehyde, was increased during H. nana challenge infection, the peak activity coinciding with the elimination of challenge larvae. Continuous administration of a NADPH oxidase inhibitor to sensitized mice interfered with the degeneration of challenge larvae. These results suggest that intestinal eosinophils may be the major contributor to oxygen radical production in response to H. nana and that reactive oxygen species may play a part of effector molecule in the resistance to reinfection with H. nana.

Ig‐Isotype Patterns of Primary and Secondary B Cell Responses to Plasmodium Chabaudi Chabaudi Correlate with IFN‐γ and IL‐4 Cytokine Production and with CD45RB Expression by CD4+ Spleen Cells

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-gamma was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL-4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while after reinfection most of the CD4+ large had a CD45RBlow phenotype.

Mice immunized by primary vaginal Candida albicans infection develop acquired vaginal mucosal immunity.

It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense mechanism against Candida infections of the vagina. However, in an estrogen-dependent murine model of experimental vaginal candidiasis, we recently showed that systemic Candida-specific Th1-type CMI induced by immunization with Candida culture filtrate antigen had no effect on vaginal Candida population levels during the course of a vaginal infection. In the present study, mice given a second vaginal inoculation in the presence of peripheral Candida-specific Th1-type CMI induced by prior vaginal infection had anamnestic-type increased delayed-type hypersensitivity (DTH) responses, concomitant with significantly fewer Candida organisms in the vagina than in primary-infected mice. In addition, organisms in secondary-infected mice were fragmented and superficial penetration into the epithelium was reduced. The systemic presence of Candida-specific T suppressor (Ts) cells that significantly suppressed the infection-derived anamnestic DTH reactivity did not abrogate the protective effect in the vagina. Additional experiments showed that vaginally immunized mice were not protected from gastrointestinal or systemic candidiasis and, in contrast to mice with a second vaginal infection, did not demonstrate anamnestic DTH reactivity. These results suggest that a moderate level of local protection against a Candida vaginal infection can be achieved by vaginal immunization but that the protective role of acquired peripheral Candida-specific Th1-type reactivity at the vaginal mucosa appears to be limited.

Studies on the comparative migration patterns of Ascaris suum larvae between primary and re-infected mice

In the present study, the effect of primary infection to reinfection with Ascaris suum larvae was experimented in mouse model. Mice were challenged with l,000 infective stage eggs of Ascaris suum. The embryonated eggs were directly introduced into stomach of mice. Reinfection was performed at 50 days after the primary infection with same method as primary infection. Mice were sacrificed 3, 5, 7, 10, 15 and 20 days after infection in both groups respectively. Larvae collected from livers and lungs with Baermann's apparatus were enumerated and measured after sacrifice. Sera of mice were also collected at same time. The results of the experiment were as follows: With antigen prepared from coelomic fluid of adult Ascaris suum and sera collected from mice before reinfection, the production of antibody in experimental mice was confirmed by the gel-diffusion technique. In the livers of reinfected mice, the larvae were recovered up to 10 days after challenge, otherwhile in the primary infected mice, the larvae were observed up to 7 days. The maximum number of larvae were observed in the lungs of primary infected mice on 10 days after inoculation. In the lungs of reinfected mice, maximum number of larvae were recovered on 7 days after, only few larvae were recovered on 10 days after reinfection. As regards the growth of the larvae, the third stage larvae, over 500 micrometer in length, appeared in livers at 5 days after reinfection, but it couldn't be found on 7 days and 10 days after challenge. The third stage larvae continuously developed were observed in lungs of mice from 5 days after reinfection. In conclusion, it was found that development of larvae in livers of immune mice were probably repressed by the immune mechanisms being rised in livers and defence mechanism is also acting by interfering with the process of larval penetration into the lung from the liver.