Infected SCID Mice Research Articles

Characterization of anti-erythrocyte and anti-platelet antibodies in hemolytic anemia and thrombocytopenia induced by Plasmodium spp. and Babesiaspp. infection in mice.

Malaria and Babesiosis are acute zoonotic disease that caused by infection with the parasite in the phylum Apicomplexa. Severe anemia and thrombocytopenia are the most common hematological complication of malaria and babesiosis. However, the mechanisms involved have not been elucidated, and only a few researches focus on the possible role of anti-erythrocyte and anti-platelet antibodies. In this study, the Plasmodium yoelii, P. chabaudi, Babesia microti and B. rodhaini infected SCID and ICR mice. The parasitemia, survival rate, platelet count, anti-platelet antibodies, and the level of IFN-γ and interleukin (IL) -10 was tested after infection. Furthermore, the P. yoelii, P. chabaudi, B. rodhaini and B. microti infected ICR mice were treated with artesunate and diminaze, the development of the anti-erythrocyte and anti-platelet antibodies in chronic stage were examined. At last, the murine red blood cell and platelet membrane proteins probed with auto-antibodies induced by P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection were characterized by proteomic analysis. The high anti-platelet and anti-erythrocyte antibodies were detected in ICR mice after P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection. Actin of murine erythrocyte and platelet is a common auto-antigen in Plasmodium and Babesia spp. infected mice. Our findings indicate that anti-erythrocyte and anti-platelet autoantibodies contribute to thrombocytopenia and anemia associated with Plasmodium spp. and Babesia spp. infection. This study will help to understand the mechanisms of malaria and babesiosis-related thrombocytopenia and hemolytic anemia.

A SCID Mouse Model To Evaluate the Efficacy of Antivirals against SARS-CoV-2 Infection.

ABSTRACTAncestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lacks the intrinsic ability to bind to the mouse ACE2 receptor, and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the Beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different wild-type (WT) mouse species. Here, we describe the establishment of a SARS-CoV-2 Beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small-molecule inhibitors. Intranasal infection of male SCID mice with 105 50% tissue culture infective doses (TCID50) of the Beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 postinfection. Treatment of infected mice with the antiviral drugs molnupiravir (200 mg/kg, twice a day [BID]) or nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 2 and 3.9 log10 TCID50/mg of tissue, respectively, and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mouse Beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2.IMPORTANCE Unlike the ancestral SARS-CoV-2 strain, the Beta (B.1.351) variant of concern has been reported to replicate to some extent in WT mice (C57BL/6 and BALB/c). We demonstrate here that infection of SCID mice with the Beta variant resulted in high viral loads in the lungs on day 3 postinfection. Treatment of infected mice with molnupiravir or nirmatrelvir for 3 consecutive days markedly reduced the infectious virus titers in the lungs and improved lung pathology. The SARS-CoV2 SCID mouse infection model, which is ideally suited for antiviral studies, offers an advantage in comparison to other SARS-CoV2 mouse models, in that there is no need for the use of mouse-adapted virus strains or genetically modified mice. Mouse models also have advantages over hamster models because (i) lower amounts of test drugs are needed, (ii) more animals can be housed in a cage, and (iii) reagents to analyze mouse samples are more readily available than those for hamsters.

PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi.

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression.

ABSTRACTThe ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Selective Upregulation of Transcripts for Six Molecules Related to T Cell Costimulation and Phagocyte Recruitment and Activation among 734 Immunity-Related Genes in the Brain during Perforin-Dependent, CD8+ T Cell-Mediated Elimination of Toxoplasma gondii Cysts.

We recently found that an invasion of CD8+ cytotoxic T cells into tissue cysts of Toxoplasma gondii initiates an elimination of the cysts in association with an accumulation of microglia and macrophages. In the present study, we compared mRNA levels for 734 immune-related genes in the brains of infected SCID mice that received perforin-sufficient or -deficient CD8+ immune T cells at 3 weeks after infection. At 7 days after the T cell transfer, mRNA levels for only six genes were identified to be greater in the recipients of the perforin-sufficient T cells than in the recipients of the perforin-deficient T cells. These six molecules included two T cell costimulatory molecules, inducible T cell costimulator receptor (ICOS) and its ligand (ICOSL); two chemokine receptors, C-X-C motif chemokine receptor 3 (CXCR3) and CXCR6; and two molecules related to an activation of microglia and macrophages, interleukin 18 receptor 1 (IL-18R1) and chitinase-like 3 (Chil3). Consistently, a marked reduction of cyst numbers and upregulation of ICOS, CXCR3, CXCR6, IL-18R1, and Chil3 mRNA levels were also detected when the perforin-sufficient CD8+ immune T cells were transferred to infected SCID mice at 6 weeks after infection, indicating that the CD8+ T cell-mediated protective immunity is capable of eliminating mature T. gondii cysts. These results together suggest that ICOS-ICOSL interactions are crucial for activating CD8+ cytotoxic immune T cells to initiate the destruction of T. gondii cysts and that CXCR3, CXCR6, and IL-18R are involved in recruitment and activation of microglia and macrophages to the T cell-attacked cysts for their elimination.IMPORTANCE T. gondii establishes a chronic infection by forming tissue cysts, which can grow into sizes greater than 50 μm in diameter as a consequence of containing hundreds to thousands of organisms surrounded by the cyst wall within infected cells. Our recent studies using murine models uncovered that CD8+ cytotoxic T cells penetrate into the cysts in a perforin-dependent manner and induce their elimination, which is accompanied with an accumulation of phagocytic cells to the T cell-attacked target. This is the first evidence of the ability of the T cells to invade into a large target for its elimination. However, the mechanisms involved in anticyst immunity remain unclear. Immune profiling analyses of 734 immune-related genes in the present study provided a valuable foundation to initiate elucidating detailed molecular mechanisms of the novel effector function of the immune system operated by perforin-mediated invasion of CD8+ T cells into large targets for their elimination.

Enhanced tuberculosis clearance through the combination treatment with recombinant adenovirus-mediated granulysin delivery

Rationale: Tuberculosis (TB) remains the leading cause of death among infectious diseases worldwide. Poor compliance of TB patients to the lengthy treatment increases the risk of relapse and leads to the emergence of multidrug-resistant and extensively drug-resistant TB (MDR-TB and XDR-TB). More effective therapies for TB are urgently needed. We hypothesized that granulysin-mediated clearance of M. tuberculosis parasited inside and outside of alveolar macrophages in presumptive infected hosts might enhance the chemotherapeutic efficacy on TB.Methods: Recombinant adenovirus type 5 (rAd5) based therapeutic vaccines rAdhGLi and rAdhGLs (rAds) were respectively developed to express intracellular and extracellular granulysin. The ex vivo bactericidal effects of rAdhGLi and rAdhGLs were evaluated on U937 and RAW264.7 cells. The efficacy of immunotherapy with both rAdhGLi and rAdhGLs on TB SCID mice, or immunotherapy combined with chemotherapy on drug-susceptible TB or MDR-TB mouse models were further evaluated.Results: rAdhGLs, as well as rAdhGLi, showed a direct bactericidal effect on extracellular or intracellular M. tuberculosis H37Rv and MDR-TB clinical strains, respectively. Immunotherapy with a dose of 109 PFU of rAdhGLi and 109 PFU of rAdhGLs demonstrated a more significant bactericidal effect on M. tuberculosis H37Rv infected SCID mice and prolonged their survival periods than rAdhGLi or rAdhGLs alone. More importantly, chemotherapy combined with rAds immunotherapy shortened the chemotherapeutic duration to 4 months on M. tuberculosis H37Rv infected mice and prevented the relapse. Combination of rAds with chemotherapy on MDR-TB mice also more significantly decreased organ bacterial load than their single use.Conclusions: Delivery of granulysin by recombinant adenovirus to the infected lung could enhance the clearance of TB in vivo and might be a promising adjunct therapeutic vaccine for TB and MDR-TB.

An immunocompromised mouse model to infect Ixodes scapularis ticks with the relapsing fever spirochete, Borrelia miyamotoi

The hard tick-borne relapsing fever spirochete, Borrelia miyamotoi, has recently gained attention as a cause of human illness, but fundamental aspects of its enzootic maintenance are still poorly understood. Challenges to experimental studies with B. miyamotoi-infected vector ticks include low prevalence of infection in field-collected ticks and seemingly inefficient horizontal transmission from infected immunocompetent rodents to feeding ticks. To reliably produce large numbers of B. miyamotoi-infected ticks in support of experimental studies, we developed an animal model where immunocompromised Mus musculus SCID mice were used as a source of B. miyamotoi-infection for larval and nymphal Ixodes scapularis ticks. Following needle inoculation with 1 × 105 spirochetes, the SCID mice developed a high spirochetemia (greater than 1 × 107 copies of B. miyamotoi purB per mL of blood) that persisted for at least 30 d after inoculation. In comparison, immunocompetent M. musculus CD-1 mice developed transient infections, detectable for only 2–8 d within the first 16 d after needle inoculation, with a brief, lower peak spirochetemia (8.5 × 104 – 5.6 × 105purB copies per mL of blood). All larval or nymphal ticks fed on infected SCID mice acquired B. miyamotoi, but frequent loss of infection during the molt led to the proportion infected ticks of the resulting nymphal or adult stages declining to 22–29%. The ticks that remained infected after the molt had well-disseminated infections which then persisted through successive life stages, including transmission to larval offspring.

GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+ T Cell Immunology in R. typhi Infection.

ABSTRACTRickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhiGFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhiGFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhiGFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhiGFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

Variable Major Proteins as Targets for Specific Antibodies against Borrelia miyamotoi.

Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern Hemisphere. No validated serologic test exists, and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 d, coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four ∼1 wk after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF.

CD8+ T cells remove cysts of Toxoplasma gondii from the brain mostly by recognizing epitopes commonly expressed by or cross-reactive between type II and type III strains of the parasite

Our previous study demonstrated that CD8+ T cells remove cysts of Toxoplasma gondii from the brain through perforin-mediated mechanisms. We here show that a transfer of CD8+ immune T cells primed with a type II or a type III strain of T. gondii both efficiently removed cysts of a type II strain from infected SCID mice, although the former tended to be slightly more efficient than the latter. Similarly, a transfer of type II-primed CD8+ T cells removed cysts of a type III strain. Therefore, CD8+ T cells are capable of removing T. gondii cysts by recognizing epitopes commonly expressed in types II and III strains or cross-reactive between these two genotypes.

Helicobacter sp. MIT 01-6451 infection during fetal and neonatal life in laboratory mice.

Helicobacter sp. MIT 01-6451 has been detected in SPF mice kept in Japan. To characterize strain MIT 01-6451, its infection route during fetal and neonatal life and effects on pregnancy were investigated using immunocompetent and immunodeficient mouse strains (BALB/c, C57BL/6, and SCID). MIT 01-6451 was detected in the uterus, vagina, and mammary glands of 50% of infected SCID mice, whereas these tissues were all negative in immunocompetent mice. No fetal infections with MIT 01-6451 were detected at 16–18 days after pregnancy in any mouse strain. In newborn mice, MIT 01-6451 was detected in intestinal tissue of C57BL/6 and SCID mice at 9–11 days after birth, but not in BALB/c mice. The IgA and IgG titers to MIT 01-6451 in sera of C57BL/6 female mice were significantly lower than those of BALB/c mice. Although no significant differences in the number of newborns per litter were observed between MIT 01-6451-infected and MIT 01-6451-free dams, the birth rate was lower in infected SCID mice than in control SCID mice. The present results indicated that MIT 01-6451 infects newborn mice after birth rather than by vertical transmission to the fetus via the placenta and that MIT 01-6451 infection shows opportunistically negative effects on the birth rate. In addition, the maternal immune response may affect infection of newborn mice with MIT 01-6451 through breast milk.

VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis.

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4(+) and CD8(+) T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

VCAM-1/α4β1 integrin interaction is important for T cell recruitment to prevent reactivation of chronic infection with Toxoplasma gondii in the brain (P3326)

Abstract T. gondii establishes a chronic infection by forming cysts preferentially in the brain, and IFN-γ production by T cells is required to maintain the latency of infection. We examined the adhesion molecules important for T cell recruitment to prevent reactivation of the infection. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of these animals as well as those of infected wild-type animals. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Immune T cells from wild-type mice were treated with anti-α4 integrin or control mAbs and transferred into infected SCID or nude mice, and animals received the same mAb every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Cerebral expression of mRNA for CD3, CD4, CD8, IFN-γ, and NOS2 (an effector molecule to inhibit T. gondii growth) was 5-34 times less in mice treated with anti-α4 integrin mAb than those treated with control mAb at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA were 17 times greater in the former than the latter at 6 days after sulfadiazine discontinuation. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for recruiting immune T cells into the brain to prevent reactivation of chronic T. gondii infection.

IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils.

BackgroundThe interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. These interactions have hindered the ability to distinguish the relative contributions of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators to tissue damage and morbidity.MethodsEncephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality. CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (WT) or IFN-γ deficient (GKO) memory CD4+ T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade.ResultsTransfer of GKO memory CD4+ T cells into infected SCID mice induced rapid mortality compared to recipients of WT memory CD4+ T cells, despite similar virus control and demyelination. In contrast to recipients of WT CD4+ T cells, extensive neutrophil infiltration and IL-17 expression within the CNS in recipients of GKO CD4+ T cells provided a model to directly assess their contribution(s) to disease. Recipients of WT CD4+ T cells depleted of IFN-γ did not express IL-17 and were spared from mortality despite abundant CNS neutrophil infiltration, indicating that mortality was not mediated by excessive CNS neutrophil accumulation. By contrast, IL-17 depletion rescued recipients of GKO CD4+ T cells from rapid mortality without diminishing neutrophils or reducing GM-CSF, associated with pathogenic Th17 cells in CNS autoimmune models. Furthermore, co-transfer of WT and GKO CD4+ T cells prolonged survival in an IFN-γ dependent manner, although IL-17 transcription was not reduced.ConclusionsThese data demonstrate that IL-17 mediates detrimental clinical consequences in an IFN-γ-deprived environment, independent of extensive neutrophil accumulation or GM-CSF upregulation. The results also suggest that IFN-γ overrides the detrimental IL-17 effector responses via a mechanism downstream of transcriptional regulation.

Animal models for Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan of worldwide distribution. This unit describes murine models of acute T. gondii infection, toxoplasmic encephalitis, and Toxoplasma retinochoroiditis. T. gondii infection in SCID mice allows the study of T cell-independent mechanisms of defense. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii allow studies of immunization and adoptive transfer. In vivo study of parasite host-interaction is possible with the use of parasites that express fluorescent proteins and model antigens, plus the use of transgenic mice that express the appropriate T cell receptor and fluorescently labeled leukocytes. Parasites that express bioluminescent markers make it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens.

Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium

We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.

Abstract 403: Deletion of HBZ alters lymphomagenesis in HTLV-1 infected humanized SCID mice

Abstract Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia (ATL), an aggressive, mature CD4+/CD25+ T-cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HPCs). Humanized NOD/SCID (HU-SCID) mice generated by injection with HTLV-1-infected CD34+ HPCs demonstrate persistent infection and reproducibly develop CD4+ T-cell lymphomas (∼80%) with clinical characteristics associated with ATL. The HTLV-1 bZIP protein (HBZ) is consistently expressed in all ATL cells and has been implicated in the maintenance of leukemogenesis. HBZ has previously been demonstrated to modulate Tax activity and to interact with the cellular factors CREB, Jun and p53. Transduction of HBZ into CD34+ HP/HSCs using a lentivirus vector suppresses clonogenic colony formation in vitro. Moreover, infection of CD34+ HP/HSCs with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ) results in elevated clonogenic colony formation in vitro, suggesting that HBZ suppresses hematopoiesis. To establish the role of HBZ in HTLV-1 replication and leukemogenesis in vivo, HU-SCID mice were infected with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ). HTLV-1ΔHBZ-infected HU-SCID mice developed lymphoproliferations that were phenotypically characterized as a mixture of immature lymphoid progenitors (CD90+) and mature B-cells (CD19+) starting at ∼10 weeks post-reconstitution, in contrast to wild-type HTLV-1 infection. Lymphoproliferations induced by HTLV-1ΔHBZ successfully engraft and expand in naïve NOD/SCID mice when injected into the peritoneal cavity and leukemic cells and lymphoma cells express HTLV-1 gp46env and p19gag. FACS-purified CD90+CD19− HTLV-1ΔHBZ infected cells also successfully engraft in naïve NOD/SCID mice and mature into CD19+ cells suggesting that these cells may be leukemic progenitors with the capability of maturing in vivo. In addition, a majority of HTLV-1ΔHBZ-infected HU-SCID mice also develop a progressive, flaccid hind limb paralysis with features similar to the neurological disorder, HAM/TSP. These results suggest that HBZ is dispensable for HTLV-1 replication and modulates lymphomagenesis in the HU-SCID model. The humanized SCID mouse provides a novel in vivo platform for analysis of the role of HTLV-1 genes in viral replication and pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 403.

Adult T-cell leukemia/lymphoma development in HTLV-1–infected humanized SCID mice

AbstractThe molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1–infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34+ hematopoietic progenitor and stem cells (CD34+ HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4+ T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34+CD38−) in the bone marrow were observed in mice developing malignancies. Purified CD34+ HP/HSCs from HTLV-1–infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow–derived stem cells. NOD/SCID mice reconstituted with CD34+ HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4+ lymphomas. The recapitulation of a CD4+ T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.

Lack of Interleukin-12 in p40-Deficient Mice Leads to Poor CD8+T-Cell Immunity againstEncephalitozoon cuniculiInfection

A CD8(+) T-cell response is critical for protection against Encephalitozoon cuniculi infection. However, the factors responsible for the generation of CD8(+) T-cell immunity during E. cuniculi infection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8(+) T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8(+) T cells from immunocompetent donors protected SCID mice infected with E. cuniculi, whereas administration of CD8(+) T cells from p40(-/-) mice failed to protect infected SCID mice. In vitro dendritic cell (DC) cultures from knockout mice pulsed with E. cuniculi spores were unable to develop a robust CD8(+) T-cell immune response. Addition of exogenous IL-12 or transfer of CD8(+) T cells that were initially primed with DC from p40(-/-) animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8(+) T-effector immunity was independent of gamma interferon (IFN-gamma) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8(+) T-cell immunity against E. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.