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A previously described heterophil degranulation assay was adapted for use with ileal mucosal tissue via quantification of β-D-glucuronidase and assay end product 4-methylumbelliferone (4-MU). Three initial experiments evaluated the effect of in ovo inoculations of Citrobacter freundii (CF) or mixed lactic acid bacteria (LAB) on ileal granulocyte degranulation. Inoculations were administered on embryonic d18, body weights (BW) were recorded on day of hatch (DOH) and d10 to calculate body weight gain (BWG), and ileal mucosal scrapings were collected on DOH or d10 for the 4-MU assay. In all experiments, treatments were statistically analyzed relative to control groups. Treatments minimally affected BWG in all in ovo experiments (p > 0.05) relative to respective control groups. Similarly, ileal degranulation in in ovo treatments did not statistically differ (p > 0.05). Based on BWG, in ovo treatments may have induced low-level inflammation unable to elicit detectable changes via the 4-MU assay. Four subsequent experiments were conducted to evaluate effects of Eimeria maxima (EM) on ileal degranulation. Treatments included non-inoculated controls and low, medium, or high EM infection. Across all four experiments, final BW or BWG over the inoculation period were suppressed (p < 0.05) in EM groups relative to respective controls with the exception of EM-low (p = 0.094) and EM-medium (p = 0.096) in one trial. Ileal mucosal scrapings for the 4-MU assay were collected on day of peak lesions. Resulting values were reduced (p < 0.05) for EM treated birds in three experiments with the exception of EM-medium (p = 0.247). No differences were observed in one experiment (p = 0.351), which may have been attributed to a variation in strain of infecting Eimeria. Although refinement for low level inflammation is warranted, results indicate successful adaptation of the 4-MU assay for use with intestinal tissue during significant gastrointestinal inflammation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Duff et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

By the last third of life, most mammals, including humans, exhibit a decline in immune cell numbers, immune organ structure, and immune defense of the organism, commonly known as immunosenescence. This decline leads to clinical manifestations of increased susceptibility to infections, particularly those caused by emerging and reemerging microorganisms, which can reach staggering levels-infection with SARS-CoV-2 has been 270-fold more lethal to older adults over 80 years of age, compared to their 18-39-year-old counterparts. However, while this would be expected to be beneficial to situations where hyporeactivity of the immune system may be desirable, this is not always the case. Here, we discuss the cellular and molecular underpinnings of immunosenescence as they pertain to outcomes of solid organ and hematopoietic transplantation., (© 2023. The Author(s), under exclusive licence to American Aging Association.)

The present study used a PCR approach to characterize prevalence of coccidial species in fecal samples obtained from 40 individual Midwestern turkey flocks to characterize distribution of species in commercial flocks. Each sample was screened for 6 prominent Eimeria species using species-specific primers and was supplemented with a primary nested-PCR approach for amplification of mitochondrial cytochrome c oxidase subunit gene I where initial sample DNA concentrations were low. All samples were positive for at least one species of Eimeria, while most presented 2 (20/40) or 3 (14/40) species in total. Prevalence across farms was primarily dominated by E. meleagrimitis (97.50%), E. adenoeides (95%), and E. gallopavonis (40%). Of the samples positive for E. adenoeides and E. meleagrimitis, almost half (17/40) contained additional species. Data presented here offer insight into Eimeria species currently challenging the Midwestern US turkey industry and potential need to evaluate flocks for species prior to implementing vaccination programs., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

Persistent inflammation has been identified as a contributor to aging-related neurodegenerative disorders such as Alzheimer's disease. Normal aging, in the absence of dementia, also results in gradual cognitive decline and is thought to arise, in part, because of a chronic pro-inflammatory state in the brain. Toxoplasma gondii is an obligate intracellular parasite that establishes a persistent, asymptomatic infection of the central nervous system (CNS) accompanied by a pro-inflammatory immune response in many of its hosts, including humans and rodents. Several studies have suggested that the inflammation generated by certain strains of T. gondii infection can be neuroprotective in the context of a secondary insult like beta-amyloid accumulation or stroke. Given these neuroprotective studies, we hypothesized that a prolonged infection with T. gondii may protect against age-associated decline in cognition. To test this hypothesis, we infected young adult mice with either of two genetically distinct, persistent T. gondii strains (Prugniaud/type II/haplogroup 2 and CEP/type III/haplogroup 3) and monitored mouse weight, survival, and learning and memory over the ensuing 20 months. At the end of the study, we evaluated CNS inflammation and parasite burden in the surviving mice. We found that parasite infection had no impact on age-associated decline in learning and memory and that by 20 months post infection, in the surviving mice, we found no evidence of parasite DNA, cysts, or inflammation in the CNS. In addition, we found that mice infected with type III parasites, which are supposed to be less virulent than the type II parasites, had a lower rate of long-term survival. Collectively, these data indicate that T. gondii may not cause a life-long CNS infection. Rather, parasites are likely slowly cleared from the CNS and infection and parasite clearance neither positively nor negatively impacts learning and memory in aging.

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum , the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum , the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei , and absent in Plasmodium yoelii In Plasmodium knowlesi , telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi , the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens., Competing Interests: The authors declare no conflict of interest.

Genetic and pathologic data suggest that amyloid beta (Aβ), produced by processing of the amyloid precursor protein, is a major initiator of Alzheimer's disease (AD). To gain new insights into Aβ modulation, we sought to harness the power of the coevolution between the neurotropic parasite Toxoplasma gondii and the mammalian brain. Two prior studies attributed Toxoplasma-associated protection against Aβ to increases in anti-inflammatory cytokines (TGF-β and IL-10) and infiltrating phagocytic monocytes. These studies only used one Toxoplasma strain making it difficult to determine if the noted changes were associated with Aβ protection or simply infection. To address this limitation, we infected a third human amyloid precursor protein AD mouse model (J20) with each of the genetically distinct, canonical strains of Toxoplasma (Type I, Type II, or Type III). We then evaluated the central nervous system (CNS) for Aβ deposition, immune cell responses, global cytokine environment, and parasite burden. We found that only Type II infection was protective against Aβ deposition despite both Type II and Type III strains establishing a chronic CNS infection and inflammatory response. Compared with uninfected and Type I-infected mice, both Type II- and Type III-infected mice showed increased numbers of CNS T cells and microglia and elevated pro-inflammatory cytokines, but neither group showed a >2-fold elevation of TGF-β or IL-10. These data suggest that we can now use our identification of protective (Type II) and nonprotective (Type III) Toxoplasma strains to determine what parasite and host factors are linked to decreased Aβ burden rather than simply with infection.

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions and in response to inflammation due to infection or stress. Whereas the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occur in the spleen during acute Plasmodium infection, a critical step in the host immune response. In this study, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a lineage(-)Sca-1(+)c-Kit(-) (LSK(-)) phenotype that proliferates in response to infection with nonlethal Plasmodium yoelii 17X. Infection-derived LSK(-) cells upon transfer into naive congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK(-) cells gave rise to B cells capable of entering into a germinal center reaction, and they developed into memory B cells and Ab-secreting cells that were capable of producing parasite-specific Abs. Differentiation of LSK(-) cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK(-) cells expand and differentiate in direct response to the parasite. However, the ability of LSK(-) cells to differentiate into B cells was not dependent on MyD88, as myd88(-/-) LSK(-) cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B lymphocytes in the spleen and are capable of contributing to the ongoing humoral immune response against Plasmodium infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection.

Neurocognition and psychopathology are robust predictors of community functioning and relapse/rehospitalization in schizophrenia. Existing studies are however limited because they have ignored the most chronic, treatment-resistant patients. Moreover, the prediction of functional outcomes has yet to be extended to the duration of community tenure, an indicator of the capacity of chronically-hospitalized patients to gain traction in the community. The current study examined neurocognition and symptom severity at discharge as potential predictors of community tenure in chronically-hospitalized treatment-resistant patients. The study recruited 90 people with treatment-resistant schizophrenia who received services on an inpatient unit. Participants completed measures of psychopathology and neurocognition prior to discharge. Following discharge, participants were tracked at current residences six months and one year post-discharge to assess community tenure. The percentage of individuals who continued to live in the community at 12-month follow-up was 51%. Severe negative symptoms but not neurocognitive impairment or positive symptoms was a significant predictor of shorter post-hospital community tenure. Of the negative symptoms domain, anhedonia-asociality proved to be the most relevant predictor of community tenure in the sample. The capacity to elicit goal-directed behaviors in response to anticipated physical and social rewards may be an important treatment target for sustaining community tenure., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Amphibian populations are declining globally. The potential contribution of glucocorticoid hormones to these declines has received little attention, but chronic elevation of glucocorticoids has been linked to a suite of negative outcomes across vertebrate taxa. Recently, chronic environmental stress has been associated with precipitous declines in sperm count and sperm viability in White's treefrogs (Litoria caerulea), but the mechanism remains unknown. In order to determine whether corticosterone is responsible for suppressing reproductive and immune function in this species, we elevated circulating concentrations of corticosterone in 10 male captive-bred frogs via transdermal application for 7 days. We compared sperm count, sperm viability, splenic cell count and circulating leucocyte counts in corticosterone-treated frogs with those in untreated control frogs. Chronic application of exogenous corticosterone led to supraphysiological circulating concentrations of corticosterone, but had no effect on sperm count or viability. However, corticosterone-treated frogs demonstrated a significant decrease in circulating eosinophils, which are immune cells implicated in fighting a variety of pathogens, including extracellular parasites. These findings suggest that although chronic elevation of circulating corticosterone is not necessarily associated with reproductive suppression in this species, it may cause immunosuppression. Thus, chronic glucocorticoid elevations in amphibians might enhance susceptibility to infection with pathogens and parasites, and their potential contributions to global population declines warrant further study.

Urbanization is a major driver of ecological change and comes with a suite of habitat modifications, including alterations to the local temperature, precipitation, light and noise regimes. Although many recent studies have investigated the behavioural and ecological ramifications of urbanization, physiological work in this area has lagged. We tested the hypothesis that anthropogenic noise is a stressor for amphibians and that chronic exposure to such noise leads to reproductive suppression. In the laboratory, we exposed male White's treefrogs, Litoria caerulea, to conspecific chorus noise either alone or coupled with pre-recorded traffic noise nightly for 1 week. Frogs presented with anthropogenic noise had significantly higher circulating concentrations of corticosterone and significantly decreased sperm count and sperm viability than did control frogs. These results suggest that in addition to having behavioural and ecological effects, anthropogenic change might alter physiology and Darwinian fitness. Future work should integrate disparate fields such as behaviour, ecology and physiology to elucidate fully organisms' responses to habitat change.

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.

In the more than 100 years since its discovery, our knowledge of Toxoplasma biology has improved enormously. The evolution of molecular biology, immunology and genomics has had profound influences on our understanding of this ubiquitous bug. However, it could be argued that in science today the adage "seeing is believing" has never been truer. Images are highly influential and in the time since the first description of T. gondii, advances in microscopy and imaging technology have been and continue to be dramatic. In this review we recount the discovery of T. gondii and the contribution of imaging techniques to elucidating its life cycle, biology and the immune response of its host.

Parasites are diverse eukaryotic pathogens that can have complex life cycles. Their clearance, or control within a mammalian host requires the coordinated effort of the immune system. The cell types recruited to areas of infection can combat the disease, promote parasite replication and survival, or contribute to disease pathology. Location and timing of cell recruitment can be crucial. In this review, we explore the role chemokines play in orchestrating and balancing the immune response to achieve optimal control of parasite replication without promoting pathology.

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

Panic is commonly co-morbid with schizophrenia. Panic may emerge prodromally, contribute to specific psychotic symptoms, and predict medication response. Panic is often missed due to agitation, impaired cognition, psychotic symptom overlap and limited clinician awareness. Carbon dioxide exposure has been used reliably to induce panic in non-psychotic panic subjects, but has not been systematically studied in schizophrenia. Eight inpatients with schizophrenia, recent auditory hallucinations, none preselected for panic, all on antipsychotic medication, received a structured Panic and Schizophrenia Interview (PaSI), assessing DSM-IV panic symptoms concurrent with paroxysmal auditory hallucinations. On that interview, all eight subjects reported panic concurrent with auditory hallucinations. At one sitting, subjects were exposed, in random order, to 35% carbon dioxide and to placebo room air, blinded to condition. All subjects experienced panic to carbon dioxide, one with limited symptoms. Only one subject panicked to placebo. One subject (one of only two without antipanic medication) had paroxysmal voices concurrent with induced panic. With added adjunctive clonazepam, that patient had marked clinical improvement and no response to carbon dioxide re-challenge. This first systematic examination offers preliminary evidence that carbon dioxide safely induces panic symptoms in schizophrenia. Panic may be prevalent and pathophysiologically significant in schizophrenia with auditory hallucinations., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)