Acute Infection In Mice Research Articles

Calreticulin (CALR) promotes ionophore-induced microneme secretion in Toxoplasma gondii.

The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER) that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly downregulated (p < 0.05). The cellular biological functions of the downregulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

Autophagy restricts Mycobacterium tuberculosis during acute infection in mice.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

Increased angiogenesis parallels cardiac tissue remodelling in experimental acute Trypanosoma cruzi infection.

Angiogenesis has been implicated in tissue injury in several noninfectious diseases, but its role in Chagas disease (CD) physiopathology is unclear. The present study aimed to investigate the effect of Trypanosoma cruzi infection on cardiac angiogenesis during the acute phase of experimental CD. The signalling pathway involved in blood vessel formation and cardiac remodelling was evaluated in Swiss Webster mice infected with the Y strain of T. cruzi. The levels of molecules involved in the regulation of angiogenesis, such as vascular endothelial growth factor-A (VEGF-A), Flk-1, phosphorylated extracellular-signal-regulated protein kinase (pERK), hypoxia-inducible factor-1α (HIF-1α), CD31, α-smooth muscle actin (α-SMA) and also the blood vessel growth were analysed during T. cruzi infection. Hearts were analysed using conventional histopathology, immunohistochemistry and western blotting. In this study, our data demonstrate that T. cruzi acute infection in mice induces exacerbated angiogenesis in the heart and parallels cardiac remodelling. In comparison with noninfected controls, the cardiac tissue of T. cruzi-infected mice presented higher levels of (i) HIF-1α, VEGF-A, Flk-1 and pERK; (ii) angiogenesis; (iii) α-SMA+ cells in the tissue; and (iv) collagen -1 deposition around blood vessels and infiltrating throughout the myocardium. We observed cardiac angiogenesis during acute experimental T. cruzi infection parallels cardiac inflammation and remodelling.

Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export.

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.

Isthmin 1 identifies a subset of lung hematopoietic stem cells and it is associated with systemic inflammation

Abstract Hematopoiesis is a highly regulated process and it’s widely described in bone marrow, the classical niche for hematopoietic stem cells (HSCs). The lungs are a novel niche of HSCs, as recently described by Lefrançais in 2017, however, little is known about the nature of these cells. Isthmin 1 (ISM1) is a secreted molecule important in developmental hematopoiesis in zebrafish; moreover, it is known that murine lungs express high levels of ISM1 and its secretion is altered after LPS challenge. In this work we performed multiparametric flow cytometry identifying a subpopulation of lung-resident HSCs expressing ISM1. Then we wanted to assess if HSCs ISM1+ cells are affected under inflammatory insults. Acute infection in mice with Pseudomonas aeruginosa showed that lung-derived HSCs ISM1+ are increased, indicating that the lung-HSCs are perturbed. Due to our model produced systemic inflammation we sought to investigate whether circulating ISM1+ cells were affected, we found that the percentage and absolute numbers of ISM1+ cells in blood were slightly diminished. Since ISM1 is a secreted molecule we evaluated its plasmatic levels by ELISA. We discovered a reduction in ISM1 plasmatic levels during murine sepsis with Pseudomonas aeruginosa. Then, we sought to analyze ISM1 levels in human inflammation; we retrieved samples from pediatric individuals with sepsis and adult patients with 2th and 3th grade burn injuries, and in both cases, we detected reduced levels of plasmatic ISM1. Our data suggest that ISM1 is likely a regulator of hematopoiesis in the lung but also may have a function in the regulation of systemic inflammation in mice and humans. We suggest that ISM1 is a novel biomarker of systemic inflammation.

Amphiphilic block copolymer delivery of a DNA vaccine against Zika virus

Zika virus (ZIKV) is a mosquito-borne flavivirus that was first discovered in 1947. Since then, outbreaks have been reported in tropical Africa, Southeast Asia, the Pacific Islands, and, in 2015, in the Americas. Since 2013, many countries have reported cases of microcephaly and other central nervous system malformation associated with ZIKV. Because the initial target population for a ZIKV vaccine is expected to be women of child-bearing age, including those who may be pregnant, it is necessary to develop safe, easily administered, and non-viral vaccines. Here, we show that a single tetrafunctional Amphiphilic Block Copolymer (ABC) delivers DNA that encodes the full natural sequence of prM-E, among other antigen designs tested, induces the highest antibody titer and neutralization activity against three divergent ZIKV isolates. Vaccination with a single tetrafunctional block copolymer delivering low dose (10 µg) DNA plasmid rapidly induces protection from detectable viremia during acute infection in mice challenged by ZIKV more than 7 months after their first vaccination and boosted 2 weeks before challenge. This use of tetrafunctional ABCs is a new approach to deliver DNA antigens against flaviviruses. The data demonstrate that DNA formulated by a tetrafunctional block copolymer rapidly elicits protective responses against multiple diverse ZIKV isolates. This represents potential for an easy-to-administer and simple to manufacture vaccine candidate against ZIKV and possibly other emerging threats to global health.

IRF9 Prevents CD8+ T Cell Exhaustion in an Extrinsic Manner during Acute Lymphocytic Choriomeningitis Virus Infection.

Effective CD8+ T cell responses play an important role in determining the course of a viral infection. Overwhelming antigen exposure can result in suboptimal CD8+ T cell responses, leading to chronic infection. This altered CD8+ T cell differentiation state, termed exhaustion, is characterized by reduced effector function, upregulation of inhibitory receptors, and altered expression of transcription factors. Prevention of overwhelming antigen exposure to limit CD8+ T cell exhaustion is of significant interest for the control of chronic infection. The transcription factor interferon regulatory factor 9 (IRF9) is a component of type I interferon (IFN-I) signaling downstream of the IFN-I receptor (IFNAR). Using acute infection of mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong, we show here that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and IFN-I and by controlling levels of IRF7, a transcription factor essential for IFN-I production. Infection of IRF9- or IFNAR-deficient mice led to a loss of early restriction of viral replication and impaired antiviral responses in dendritic cells, resulting in CD8+ T cell exhaustion and chronic infection. Differences in the antiviral activities of IRF9- and IFNAR-deficient mice and dendritic cells provided further evidence of IRF9-independent IFN-I signaling. Thus, our findings illustrate a CD8+ T cell-extrinsic function for IRF9, as a signaling factor downstream of IFNAR, in preventing overwhelming antigen exposure resulting in CD8+ T cell exhaustion and, ultimately, chronic infection.IMPORTANCE During early viral infection, overwhelming antigen exposure can cause functional exhaustion of CD8+ T cells and lead to chronic infection. Here we show that the transcription factor interferon regulatory factor 9 (IRF9) plays a decisive role in preventing CD8+ T cell exhaustion. Using acute infection of mice with LCMV strain Armstrong, we found that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and Irf7, encoding a transcription factor crucial for type I interferon (IFN-I) production, as well as by controlling the levels of IFN-I. Infection of IRF9-deficient mice led to a chronic infection that was accompanied by CD8+ T cell exhaustion due to defects extrinsic to T cells. Our findings illustrate an essential role for IRF9, as a mediator downstream of IFNAR, in preventing overwhelming antigen exposure causing CD8+ T cell exhaustion and leading to chronic viral infection.

LVS vaccination allows BALB/c mice to survive virulent F. tularensis pulmonary challenge but does not induce sterilizing immunity

Abstract There is no licensed vaccine for protection against virulent strains of Francisella tularensis, an intracellular pathogen that replicates within lung macrophages and causes lethal acute infection in mice at an intranasal challenge dose of ~10 CFU. Immunization with the attenuated F. tularensis LVS strain provides some level of protection against virulent F. tularensis SchuS4 infection; however, the basis for this induced resistance remains unknown. We found that LVS vaccination allowed complete survival of BALB/c mice (but not C57Bl/6 mice) from lethal SchuS4 pulmonary challenge for 30 days, and 50% survival for at least 100 days, yet the surviving mice contained large numbers of SchuS4 organisms in their lungs and spleens (~104 CFU/organ). Early after challenge, resistant BALB/c mice produced greater amounts of Th17-associated cytokines compared to susceptible C57Bl/6 mice. However, IL-17 and IL-22 did not appear to be important for restricting chronic infection. On the other hand, depletion of either CD4+ or CD8+ cells three weeks after SchuS4 challenge caused resistant BALB/c mice to succumb to infection within one week. Similarly, treatment of vaccinated BALB/c mice with anti-IFNγ monoclonal antibodies three weeks after SchuS4 led to fatal acute tularemia. We conclude that LVS vaccination of BALB/c mice induces an immune response that allows the animals to constrain and tolerate a chronic SchuS4 infection. Long-term resistance is dependent on CD4+ cells, CD8+ cells, and IFNγ.

A Herpes Simplex Virus 2 (HSV-2) gD Mutant Impaired for Neural Tropism Is Superior to an HSV-2 gD Subunit Vaccine To Protect Animals from Challenge with HSV-2.

A recent phase 3 trial with soluble herpes simplex virus 2 (HSV-2) glycoprotein D (gD2t) in adjuvant failed to show protection against genital herpes. We postulated that live attenuated HSV-2 would provide more HSV antigens for induction of virus-specific antibodies and cellular immunity than would gD2t. We previously reported an HSV-2 mutant, HSV2-gD27, in which the nectin-1 binding domain of gD2 is altered so that the virus is impaired for infecting neural cells, but not epithelial cells, in vitro and is impaired for infecting dorsal root ganglia in mice (K. Wang, J. D. Kappel, C. Canders, W. F. Davila, D. Sayre, M. Chavez, L. Pesnicak, and J. I. Cohen, J Virol 86:12891-12902, 2012, doi:10.1128/JVI.01055-12). Here we report that the mutations in HSV2-gD27 were stable when the virus was passaged in cell culture and during acute infection of mice. HSV2-gD27 was attenuated in mice when it was inoculated onto the cornea, intramuscularly (i.m.), intravaginally, and intracranially. Vaccination of mice i.m. with HSV2-gD27 provided better inhibition of challenge virus replication in the vagina than when the virus was used to vaccinate mice intranasally or subcutaneously. Comparison of i.m. vaccinations with HSV2-gD27 versus gD2t in adjuvant showed that HSV2-gD27 induced larger reductions of challenge virus replication in the vagina and reduced latent viral loads in dorsal root ganglia but induced lower serum neutralizing antibody titers than those obtained with gD2t in adjuvant. Taken together, our data indicate that a live attenuated HSV-2 vaccine impaired for infection of neurons provides better protection from vaginal challenge with HSV-2 than that obtained with a subunit vaccine, despite inducing lower titers of HSV-2 neutralizing antibodies in the serum. Genital herpes simplex is one of the most prevalent sexually transmitted diseases. Though HSV-2 disease is usually mild, it can be life threatening in neonates and immunocompromised persons. In addition, genital herpes increases the frequency of HIV infection and transmission. HSV-2 maintains a latent infection in sensory neurons and cannot be cleared with antiviral drugs. The virus frequently reactivates, resulting in virus shedding in the genital area, which serves as a source for transmission. A prophylactic vaccine is needed to prevent disease and to control the spread of the virus. Previous human trials of subunit vaccines have been unsuccessful. Here we report the results of vaccinating mice with a new type of live attenuated HSV-2 vaccine that is impaired for infection of neurons and provides better protection of mice than that obtained with a subunit vaccine. The strategy of altering the cell tropism of a virus is a new approach for a live attenuated vaccine.

Immunology: Chronic effects of acute infections.

Acute infection of mice with an intestinal pathogen leads to long-lasting inflammation that is maintained by intestinal microorganisms. This observation reveals a path by which infection history can affect long-term immune function.

The conspiracy of autophagy, stress and inflammation in acute pancreatitis.

Acute pancreatitis is associated with alcohol abuse, gallstones and bacterial infection. Its basic cause is tissue destruction accompanied by an innate immune response, which induces epithelial stress pathways. Recent studies have focused on some of the integral cellular pathways shared between multiple pancreatitis models that also suggest new approaches to detection and treatment. Several models of pancreatitis have been associated with stress responses, such as endoplasmic reticulum and oxidative stress together with the induction of a defective autophagic pathway. Recent evidence reinforces the critical role of these cellular processes in pancreatitis. A member of the toll-like receptor family, toll-like receptor 4, which is known to contribute to disease pathology in many models of experimental pancreatitis, has been found to be a promising target for treatment of pancreatitis. Interestingly, a direct activator of toll-like receptor 4, the bacterial cell wall component in gram-negative bacteria lipopolysaccharide, contributes to the onset and severity of disease when combined with additional stressors, such as chronic alcohol feeding; however, recent studies have shown that acute infection of mice with live bacteria is alone sufficient to induce acute pancreatitis. In the last several months, the convergent roles of acinar cell stress, autophagy and proinflammatory signaling initiated by the toll-like receptors have been emphatically reinforced in the onset of acute pancreatitis.

Chronic infection with trypanosoma cruzi alters the phenotypic profile of circulating memory B cells in humans (MPF2P.812)

Abstract Trypanosoma cruzi was shown to induce the loss of parasite-specific IgG-producing B cells during the acute infection of mice, which was proposed to hamper B cell responses and favor parasite replication and chronicity (Zuñiga et al. 2002). Herein, we measured the levels of circulating memory and MZ-like B cells in 12 individuals chronically infected with T. cruzi and 9 healthy controls based on the expression of CD19, IgM IgD, IgG, and CD27. T. cruzi-infected individuals had significantly lower percentages of IgM+IgD-CD27+ (IgM-only), IgM-IgD-CD27+ (isotype-switched) and IgG+IgD-CD27+ memory B cells compared to controls (p&amp;lt;0.05). Also, the percentage of IgM+IgD+CD27+ (MZ-like) B cells was reduced in infected individuals while the proportion of IgG+IgD-CD27- memory B cells was increased (p&amp;lt;0.05). The levels of memory B cells were further analyzed according to ECG findings in infected individuals (asymptomatic [ASY] and chronic Chagas heart disease [CHD]). IgG+IgD-CD27+, IgM+IgD-CD27+ and IgM-IgD-CD27+ B cells were significantly reduced in the ASY group compared to controls (p&amp;lt;0.05); CHD individuals had decreased levels of IgM-IgD-CD27+ and IgM+IgD-CD27+ B cells (p&amp;lt;0.05). Altogether, our data indicate that chronic exposure to T. cruzi modulates the phenotypic profile of the peripheral blood CD19+ B cell compartment in humans, and that some of the defects develop early in the infection, prior the onset of heart symptoms. Supported by Argentinian MINCYT/FONCYT PICT 2010-2148

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3−/− mice but increased tissue damage when given to WT mice.

Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence

The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp(55) (W55) or Gly(57) (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe(50) (F50) and Lys(62) (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe(5) (F5) and Lys(41) (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.

Acute infection of mice with Clostridium difficile leads to eIF2α phosphorylation and pro‐survival signalling as part of the mucosal inflammatory response

The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C.difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C.difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C.difficile and highlight eIF2α phosphorylation and the interleukin-22-pSTAT3-RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.

Anemia Associated With Acute Infection in Children

We constructed an animal model to examine the possibility that erythrophagocytosis may contribute to decreased hemoglobin (Hgb) levels in acute infection in mice. BALB/c mice weighing 20 to 25 g were injected (intraperitoneally) with lipopolysaccharide (LPS) (Escherichia coli serotype) of some concentrations. Control mice were injected intraperitoneally with saline (0.5 mL). Two and 4 hours after LPS administration, mice were bled (0.25 mL) for complete blood count measures and tumor necrosis factor-α and interleukin-6 levels. The mice were then killed, and their spleen, liver, and bone marrow were examined microscopically for erythrophagocytosis. After LPS administration, mouse Hgb and hematocrit levels dropped significantly. At 4 hours after LPS injection, all Hgb and hematocrit concentrations were found to be significantly lower compared with that of controls (P = 0.002 and 0.001, respectively). Significantly increased concentrations of tumor necrosis factor-α and interleukin-6 were evident after LPS injection. Prominent hepatic erythrophagocytosis was observed in the LPS-injected mice compared with controls. A significant across-group difference was observed at 4 hours, driven by significantly higher values in group 500 mcg versus controls (P = 0.005) and 100 mcg (P = 0.025). A significant increase in erythrophagocytes was observed at 2 to 4 hours in the 500 mcg LPS group (P = 0.044). Erythrophagocytosis may play a role in anemia associated with acute infection in mice.

IFN-γ from CD4 T Cells Is Essential for Host Survival and Enhances CD8 T Cell Function during Mycobacterium tuberculosis Infection

IFN-γ is necessary in both humans and mice for control of Mycobacterium tuberculosis. CD4 T cells are a significant source of IFN-γ during acute infection in mice and are required for control of bacterial growth and host survival. However, several other types of cells can and do produce IFN-γ during the course of the infection. We sought to determine whether IFN-γ from sources other than CD4 T cells was sufficient to control M. tuberculosis infection and whether CD4 T cells had a role in addition to IFN-γ production. To investigate the role of IFN-γ from CD4 T cells, a murine adoptive transfer model was developed in which all cells were capable of producing IFN-γ, with the exception of CD4 T cells. Our data in this system support that CD4 T cells are essential for control of infection, but also that IFN-γ from CD4 T cells is necessary for host survival and optimal long-term control of bacterial burden. In addition, IFN-γ from CD4 T cells was required for a robust CD8 T cell response. IFN-γ from T cells inhibited intracellular replication of M. tuberculosis in macrophages, suggesting IFN-γ may be necessary for intracellular bactericidal activity. Thus, although CD4 T cells play additional roles in the control of M. tuberculosis infection, IFN-γ is a major function by which these cells participate in resistance to tuberculosis.

Trypanosoma cruzi Entrance through Systemic or Mucosal Infection Sites Differentially Modulates Regional Immune Response Following Acute Infection in Mice

Acute Chagas disease is characterized by a systemic infection that leads to the strong activation of the adaptive immune response. Outbreaks of oral contamination by the infective protozoan Trypanosoma cruzi are frequent in Brazil and other Latin American countries, and an increased severity of clinical manifestations and mortality is observed in infected patients. These findings have elicited questions about the specific responses triggered after T. cruzi entry via mucosal sites, possibly modulating local immune mechanisms, and further impacting regional and systemic immunity. Here, we provide evidence for the existence of differential lymphoid organ responses in experimental models of acute T. cruzi infection.

In vivo activity of thiophene-containing trisubstituted methanes against acute and persistent infection of non-tubercular Mycobacterium fortuitum in a murine infection model

Mycobacterium fortuitum causes opportunist non-tubercular infection in humans. Chronic infection of M. fortuitum has been clinically documented and requires prolonged chemotherapy. The objectives of this study were to characterize acute and persistent infection of M. fortuitum in a murine infection model and to screen thiophene-containing trisubstituted methanes active against both acute and persistent infection. A murine infection model of M. fortuitum was used. Bacillary count, bioluminescence, disease symptoms, host immune response, drug susceptibility and mortality were measured. Reactivation of persistent bacilli was induced by dexamethasone. Trisubstituted methanes containing thiophene rings were synthesized and screened in vitro by agar dilution and BACTEC assay and in mice. Cytotoxicity was tested with Vero monkey kidney cells using a resazurin assay. The acute infection in mice was marked by a 3 log rise in viable counts, the appearance of disease symptoms and a rise in the Th1 immune response. Bacilli were susceptible to fluoroquinolones. This was followed by persistent infection, in which disappearance of disease symptoms, a decline in Th1 response and non-susceptibility to fluoroquinolones was observed. When the mice were immunocompromised on day 40 post-infection (persistent state) by dexamethasone, a rise in viable counts, symptoms and susceptibility to fluoroquinolones and a prominent Th1 response reappeared. Two lead compounds were found that cleared the mice of bacilli in acute infection and caused a 2.29-2.99 log reduction in cfu of persistent bacilli. The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.