Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole sporozoite PfSPZ Vaccine in African infants. Innate immune activation and myeloid signatures at pre-vaccination baseline correlated with protection from Pf parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ Vaccine dose. Machine learning identified spliceosome, proteosome, and resting dendritic cell signatures as pre-vaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline CSP-specific IgG predicted non-protection. Pre-vaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T-cell responses post-vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naïve mice while diminishing the CD8+ T-cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity of whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggest that PfSPZ Vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

Creation of a non-Western humanized gnotobiotic mouse model through the transplantation of rural African fecal microbiota.

IMPORTANCE: There is increasing evidence that microbes residing within the intestines (gut microbiota) play important roles in the well-being of humans. Yet, there are considerable challenges in determining the specific role of gut microbiota in human diseases owing to the complexity of diverse internal and environmental factors that can contribute to diseases. Mice devoid of all microorganisms (germ-free mice) can be colonized with human stool samples to examine the specific contribution of the gut microbiota to a disease. These approaches have been primarily focused on stool samples obtained from individuals in Western countries. Thus, there is limited understanding as to whether the same methods used to colonize germ-free mice with stool from Western individuals would apply to the colonization of germ-free mice with stool from non-Western individuals. Here, we report the results from colonizing germ-free mice with stool samples of Malian children.

Validated graft-specific biomarkers identify patients at risk for chronic graft-versus-host disease and death.

BACKGROUNDChronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic hematopoietic cell transplantation (HCT). More accurate information regarding the risk of developing cGVHD is required. Bone marrow (BM) grafts contribute to lower cGVHD, which creates a dispute over whether risk biomarker scores should be used for peripheral blood (PB) and BM.METHODSDay 90 plasma proteomics from PB and BM recipients developing cGVHD revealed 5 risk markers that were added to 8 previous cGVHD markers to screen 982 HCT samples of 2 multicenter Blood and Marrow Transplant Clinical Trials Network (BMTCTN) cohorts. Each marker was tested for its association with cause-specific hazard ratios (HRs) of cGVHD using Cox-proportional-hazards models. We paired these clinical studies with biomarker measurements in a mouse model of cGVHD.RESULTSSpearman correlations between DKK3 and MMP3 were significant in both cohorts. In BMTCTN 0201 multivariate analyses, PB recipients with 1-log increase in CXCL9 and DKK3 were 1.3 times (95% CI: 1.1-1.4, P = 0.001) and 1.9 times (95%CI: 1.1-3.2, P = 0.019) and BM recipients with 1-log increase in CXCL10 and MMP3 were 1.3 times (95%CI: 1.0-1.6, P = 0.018 and P = 0.023) more likely to develop cGVHD. In BMTCTN 1202, PB patients with high CXCL9 and MMP3 were 1.1 times (95%CI: 1.0-1.2, P = 0.037) and 1.2 times (95%CI: 1.0-1.3, P = 0.009) more likely to develop cGVHD. PB patients with high biomarkers had increased likelihood to develop cGVHD in both cohorts (22%-32% versus 8%-12%, P = 0.002 and P < 0.001, respectively). Mice showed elevated circulating biomarkers before the signs of cGVHD.CONCLUSIONBiomarker levels at 3 months after HCT identify patients at risk for cGVHD occurrence.FUNDINGNIH grants R01CA168814, R21HL139934, P01CA158505, T32AI007313, and R01CA264921.

A biomarker panel for risk of early respiratory failure following hematopoietic cell transplantation.

Plasma biomarkers associated with respiratory failure (RF) following hematopoietic cell transplantation (HCT) have not been identified. Therefore, we aimed to validate early (7 and 14 days post-HCT) risk biomarkers for RF. Using tandem mass spectrometry, we compared plasma obtained at day 14 post-HCT from 15 patients with RF and 15 patients without RF. Six candidate proteins, from this discovery cohort or identified in the literature, were measured by enzyme-linked immunosorbent assay in day-7 and day-14 post-HCT samples from the training (n = 213) and validation (n = 119) cohorts. Cox proportional-hazard analyses with biomarkers dichotomized by Youden's index, as well as landmark analyses to determine the association between biomarkers and RF, were performed. Of the 6 markers, Stimulation-2 (ST2), WAP 4-disulfide core domain protein 2 (WFDC2), interleukin-6 (IL-6), and tumor necrosis factor receptor 1 (TNFR1), measured at day 14 post-HCT, had the most significant association with an increased risk for RF in the training cohort (ST2: hazard ratio [HR], 4.5, P = .004; WFDC2: HR, 4.2, P = .010; IL-6: HR, 6.9, P < .001; and TFNR1: HR, 6.1, P < .001) and in the validation cohort (ST2: HR, 23.2, P = .013; WFDC2: HR, 18.2, P = .019; IL-6: HR, 12.2, P = .014; and TFNR1: HR, 16.1, P = .001) after adjusting for the conditioning regimen. Using cause-specific landmark analyses, including days 7 and 14, high plasma levels of ST2, WFDC2, IL-6, and TNFR1 were associated with an increased HR for RF in the training and validation cohorts. These biomarkers were also predictive of mortality from RF. ST2, WFDC2, IL-6 and TNFR1 levels measured early posttransplantation improve risk stratification for RF and its related mortality.

Orexin signaling in rostral lateral hypothalamus and nucleus accumbens shell in the control of spontaneous physical activity in high- and low-activity rats.

Spontaneous physical activity (SPA) describes activity outside of formal exercise and shows large interindividual variability. The hypothalamic orexin/hypocretin peptides are key regulators of SPA. Orexins drive SPA within multiple brain sites, including rostral lateral hypothalamus (LH) and nucleus accumbens shell (NAcSh). Rats with high basal SPA (high activity, HA) show higher orexin mRNA expression and SPA after injection of orexin-A in rostral LH compared with low-activity (LA) rats. Here, we explored the contribution of orexin signaling in rostral LH and NAcSh to the HA/LA phenotype. We found that HA rats have higher sensitivity to SPA after injection of orexin-A in rostral LH, but not in NAcSh. HA and LA rats showed similar levels of orexin receptor expression in rostral LH, and activation of orexin-producing neurons after orexin-A injection in rostral LH. Also, in HA and LA rats, the coinjection of orexin-A in rostral LH and NAcSh failed to further increase SPA beyond the effects of orexin-A in rostral LH. Pretreatment with muscimol, a GABAA receptor agonist, in NAcSh potentiated SPA produced by orexin-A injection in rostral LH in HA but not in LA rats. Our results suggest that a feedback loop from orexin-responsive neurons in rostral LH to orexin neurons and a the NAcSh-orexin neuron-rostral LH circuit regulate SPA. Overall, our data suggest that differences in orexin sensitivity in rostral LH and its modulation by GABA afferents from NAcSh contribute to individual SPA differences.

Exercise reduces diet-induced cognitive decline and increases hippocampal brain-derived neurotrophic factor in CA3 neurons.

BACKGROUND: Previous studies have shown that a western diet impairs, whereas physical exercise enhances hippocampus-dependent learning and memory. Both diet and exercise influence expression of hippocampal brain-derived neurotrophic factor (BDNF), which is associated with improved cognition. We hypothesized that exercise reverses diet-induced cognitive decline while increasing hippocampal BDNF. METHODS: To test the effects of exercise on hippocampal-dependent memory, we compared cognitive scores of Sprague-Dawley rats exercised by voluntary running wheel (RW) access or forced treadmill (TM) to sedentary (Sed) animals. Memory was tested by two-way active avoidance test (TWAA), in which animals are exposed to a brief shock in a specific chamber area. When an animal avoids, escapes or has reduced latency to do either, this is considered a measure of memory. In a second experiment, rats were fed either a high-fat diet or control diet for 16 weeks, then randomly assigned to running wheel access or sedentary condition, and TWAA memory was tested once a week for 7 weeks of exercise intervention. RESULTS: Both groups of exercised animals had improved memory as indicated by reduced latency to avoid and escape shock, and increased avoid and escape episodes (p<0.05). Exposure to a high-fat diet resulted in poor performance during both the acquisition and retrieval phases of the memory test as compared to controls. Exercise reversed high-fat diet-induced memory impairment, and increased brain-derived neurotrophic factor (BDNF) in neurons of the hippocampal CA3 region. CONCLUSIONS: These data suggest that exercise improves memory retrieval, particularly with respect to avoiding aversive stimuli, and may be beneficial in protecting against diet induced cognitive decline, likely via elevated BDNF in neurons of the CA3 region.

Altered protein conformation and lower stability of the dystrophic transforming growth factor beta-induced protein mutants.

PURPOSE: Transforming growth factor beta-induced protein (TGFBIp) is a widely expressed extracellular matrix protein that plays roles in cell adhesion and migration, differentiation, apoptosis, bone morphogenesis, and carcinogenesis. Mutations of TGFBIp have been linked to stromal corneal dystrophies, a group of protein conformational diseases characterized by abnormal protein aggregations in the cornea. However, the underlying pathogenic mechanism remains elusive due to a lack of insight into the molecular properties of the disease-causing mutants. In the current study, we applied spectroscopic tools to compare the conformation and protein stability of recombinant wild-type (WT) TGFBIp to two dystrophic mutants, R124C and R555W. METHODS: A serum-free expression system was used to produce the recombinant TGFBIp proteins. Fluorescence and far-ultraviolet circular dichroism spectroscopies were used to compare WT and dystrophic mutants under various conditions. RESULTS: Our results showed that dystrophic mutants were processed differentially by the expressing cells and produced different proteolytic fragment patterns by proteolysis. Intrinsic tryptophan fluorescence studies revealed moderate shifts in the emission maxima and increased quenching by iodide ion of mutant TGFBIp, suggesting a different conformation than WT protein. Denaturation experiments indicated a difference in protein stability between WT and mutant proteins. Under oxidizing conditions, the mutants produced higher 1-anilinonaphthalene-8-sulfonic acid and thioflavin T fluorescence signals than the WT, indicating increased protein unfolding and fibril formation, respectively. Finally, far-ultraviolet circular dichroism spectroscopy revealed that WT TGFBIp undergoes concentration-dependent conformational changes; similar experiments were not possible on mutant TGFBIp, which remained soluble only at low concentrations. CONCLUSIONS: Our study provides new evidence for the pathogenic mechanism of dystrophic mutants. Although mutant TGFBIp has moderate but consistent structural perturbations, other factors such as oxidation or degradation may be required to cause the phenotypic abnormal aggregations.

Memory T cells persisting in the brain following MCMV infection induce long-term microglial activation via interferon-γ.

Murine cytomegalovirus (MCMV) brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. Here, we show that in response to MCMV brain infection, antigen-specific CD8(+) T cells migrated into the brain and persisted as long-lived memory cells. The role of these persistent T cells in the brain is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Strikingly, memory T cells isolated from the brain exhibited an effector phenotype and produced IFN-γ upon restimulation with viral peptide. Furthermore, we observed time-dependent and long-term activation of resident microglia, indicated by chronic MHC class II up-regulation and TNF-α production. The immune response in this immunologically restricted site persisted in the absence of active viral replication. Lymphocyte infiltrates were detected until 30 days post-infection (p.i.), with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. We then investigated the role of IFN-γ in chronic microglial activation by using IFN-γ-knockout (GKO) mice. At 30 days p.i., GKO mice demonstrated a similar phenotypic brain infiltrate when compared to wild-type mice (Wt), however, MHC class II expression on microglia isolated from these GKO mice was significantly lower compared to Wt animals. When IFN-γ producing CD8(+) T cells were reconstituted in GKO mice, MHC class II up-regulation on microglial cells was restored. Taken together, these results suggest that MCMV brain infection results in long-term persistence of antigen-specific CD8(+) T cells which produce IFN-γ and drive chronic microglial cell activation. This response was found to be dependent on IFN-γ production by viral Ag-specific T cells during the chronic phase of disease.

Excess neutrophil infiltration during cytomegalovirus brain infection of interleukin-10-deficient mice.

Wild-type mice control murine cytomegalovirus (MCMV) brain infection, but identical infection is lethal to animals deficient in interleukin (IL)-10. Here, we report that MCMV-infected IL-10 knockout (KO) mice displayed a marked increase in neutrophil infiltration into the infected, IL-10-deficient brain when compared to wild-type animals. Enhanced microglial cell activation, determined by MHC class II up-regulation, overexpression of CXCL2, and elevated P-selectin mRNA levels were observed. In vivo blocking of CXCL2 attenuated neutrophil infiltration and significantly improved the outcome of infection. Collectively, these data indicate that the absence of IL-10 results in pathologic neutrophil infiltration into MCMV-infected brains.

Herpes simplex virus induces neural oxidative damage via microglial cell Toll-like receptor-2.

BACKGROUND: Using a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis. METHODS: Purified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2-/- mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from beta-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia. RESULTS: Stimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2-/- microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2-/- microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2-/- microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2-/- mice. CONCLUSIONS: These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.

Chronic cortical and subcortical pathology with associated neurological deficits ensuing experimental herpes encephalitis.

Long-term neurological sequela is common among herpes simplex encephalitis (HSE) survivors. Animal models for HSE are used to investigate mechanisms of acute disease, but little has been done to model chronic manifestations of HSE. The current study presents a detailed, systematic analysis of chronic neuropathology, including characterization of topography and sequential progression of degenerative lesions and inflammation. Subsequent to intranasal HSV-1 infection, inflammatory responses that were temporally and spatially distinct persisted in infected cortical and brain stem regions. Neutrophils were present exclusively within the olfactory bulb and brain stem regions during the acute phase of infection, while the chronic inflammation was marked by plasma cells, lymphocytes and activated microglia. The chronic lymphocytic infiltrate, cytokine production, and activated microglia were associated with the loss of cortical neuropile in the entorhinal cortex and hippocampus. Animals surviving the acute infection showed a spectrum of chronic lesions from decreased brain volume, neuronal loss, activated astrocytes, and glial scar formation to severe atrophy and cavitations of the cortex. These lesions were also associated with severe spatial memory deficits in surviving animals. Taken together, this model can be utilized to further investigate the mechanisms of neurological defects that follow in the wake of HSE.

Denaturation and solvent effect on the conformation and fibril formation of TGFBIp.

PURPOSE: Transforming growth factor beta-induced protein (TGFBIp) aggregates into the phenotypic amyloid fibrils and/or non-amyloid deposits in corneal dystrophies and other disorders. While significant progress has been made in molecular genetics to successfully establish the link between the missense mutations of TGFBI and TGFBIp-related corneal dystrophies, the underlying mechanism for the abnormal aggregation remains elusive due to the lack of insights into the conformational perturbations induced by mutations. In the present study, we examined the effects of denaturants and a co-solvent on recombinant TGFBIp, with a focus on protein conformational changes and amyloid fibril formation. METHODS: Recombinant TGFBIp was subjected to various spectroscopic studies, such as far-ultraviolet circular dichroism (far-UV CD), intrinsic tryptophan fluorescence and quenching, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, under various denaturing conditions (urea and guanidine hydrochloride [GndHCl], acidic pH, and trifluoroethanol [TFE, co-solvent]). A thioflavin T (ThT) fluorescence assay was used to determine the fibril formation of TGFBIp. In addition, a rabbit polyclonal antibody against the oligomer precursors that initiate the formation of amyloid fibrils was also used in dot blot experiments to detect the formation of prefibrillar precursors. RESULTS: The purified recombinant TGFBIp is in the folded state according to its intrinsic tryptophan fluorescence analyses. A single-step unfolding process was observed in the GndHCl denaturation experiment. Results from far-UV CD, intrinsic tryptophan fluorescence, and ANS fluorescence experiments showed that TFE exerted its solvent effects by initially unfolding and transforming TGFBIp to a beta-sheet-enriched conformer at 20%. When increased to 40%, TFE changed TGFBIp into a non-native alpha-helix conformer. Although GndHCl and TFE led to protein unfolding, enhanced fibril formation could only be observed in the presence of TFE and at acidic pH, according to the ThT fluorescence assays. The paradigmatic protofibrillar TGFBIp oligomers were also detected during the fibril formation by the dot blot experiment. CONCLUSIONS: Our results suggest that protein unfolding may serve as the prerequisite but is not sufficient for the fibrillogenesis. Other factors, such as the solvent used, fragmentation, or pH, may also be crucial for the formation of TGFBIp fibrils.