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Background: The emergency personnel who responded to the World Trade Center (WTC) attacks endured severe occupational exposures, yet the prevalence of cognitive impairment remains unknown among WTC-exposed-FDNY-responders. The present study screened for mild and severe cognitive impairment in WTC-exposed FDNY responders using objective tests, compared prevalence rates to a cohort of non-FDNY WTC-exposed responders, and descriptively to meta-analytic estimates of MCI from global, community, and clinical populations., Methods: A sample of WTC-exposed-FDNY responders ( n = 343) was recruited to complete an extensive battery of cognitive, psychological, and physical tests. The prevalences of domain-specific impairments were estimated based on the results of norm-referenced tests, and the Montreal Cognitive Assessment (MoCA), Jak/Bondi criteria, Petersen criteria, and the National Institute on Aging and Alzheimer's Association (NIA-AA) criteria were used to diagnose MCI. NIA-AA criteria were also used to diagnose severe cognitive impairment. Generalized linear models were used to compare prevalence estimates of cognitive impairment to a large sample of WTC-exposed-non-FDNY responders from the General Responder Cohort (GRC; n = 7102) who completed the MoCA during a similar time frame., Result: Among FDNY responders under 65 years, the unadjusted prevalence of MCI varied from 52.57% to 71.37% depending on the operational definition of MCI, apart from using a conservative cut-off applied to MoCA total scores (18 < MoCA < 23), which yielded a markedly lower crude prevalence (24.31%) compared to alternative criteria. The prevalence of MCI was higher among WTC-exposed-FDNY-responders, compared to WTC-exposed-non-FDNY-GRC-responders (adjusted RR = 1.53, 95% C.I . = [1.24, 1.88], p < .001) and meta-analytic estimates from different global, community, and clinical populations. Following NIA-AA diagnostic guidelines, 4.96% of WTC-exposed-FDNY-responders met the criteria for severe impairments (95% CI = [2.91% to 7.82%]), a prevalence that remained largely unchanged after excluding responders over the age of 65 years., Discussion: There is a high prevalence of mild and severe cognitive impairment among WTC-responders highlighting the putative role of occupational/environmental and disaster-related exposures in the etiology of accelerated cognitive decline., Competing Interests: Statement of Competing Interests. The authors have no competing interests to report.

Purpose: World Trade Center (WTC) exposure is associated with obstructive airway diseases and sarcoidosis. There is limited research regarding the incidence and progression of non-sarcoidosis interstitial lung diseases (ILD) after WTC-exposure. ILD encompasses parenchymal diseases which may lead to progressive pulmonary fibrosis (PPF). We used the Fire Department of the City of New York's (FDNY's) WTC Health Program cohort to estimate ILD incidence and progression., Methods: This longitudinal study included 14,525 responders without ILD prior to 9/11/2001. ILD incidence and prevalence were estimated and standardized to the US 2014 population. Poisson regression modeled risk factors, including WTC-exposure and forced vital capacity (FVC), associated with ILD. Follow-up time ended at the earliest of incident diagnosis, end of study period/case ascertainment, transplant or death., Results: ILD developed in 80/14,525 FDNY WTC responders. Age, smoking, and gastroesophageal reflux disease (GERD) prior to diagnosis were associated with incident ILD, though FVC was not. PPF developed in 40/80 ILD cases. Among the 80 cases, the average follow-up time after ILD diagnosis was 8.5 years with the majority of deaths occurring among those with PPF (PPF: n = 13; ILD without PPF: n = 6)., Conclusions: The prevalence of post-9/11 ILD was more than two-fold greater than the general population. An exposure-response gradient could not be demonstrated. Half the ILD cases developed PPF, higher than previously reported. Age, smoking, and GERD were risk factors for ILD and PPF, while lung function was not. This may indicate that lung function measured after respirable exposures would not identify those at risk for ILD or PPF., (© 2024. The Author(s).)

Evidence suggests that reductions in healthcare utilization, including forgone care, during the COVID-19 pandemic may be contributing towards excess morbidity and mortality. The objective of this study was to describe individual and community-level correlates of forgone care during the COVID-19 pandemic. We conducted a cross-sectional, secondary data analysis of participants (n = 2,003) who reported needing healthcare in two population-representative surveys conducted in Baltimore, MD in 2021 and 2021-2022. Abstracted data included the experience of forgone care, socio-demographic data, comorbidities, financial strain, and community of residence. Participant's community of residence were linked with data acquired from the Baltimore Neighborhood Indicators Alliance relevant to healthcare access and utilization, including walkability and internet access, among others. The data were analyzed using weighted random effects logistic regression. Individual-level factors found to be associated with increased odds for forgone care included individuals age 35-49 (compared to 18-34), female sex, experiencing housing insecurity during the pandemic, and the presence of functional limitations and mental illness. Black/African American individuals were found to have reduced odds of forgone care, compared to any other race. No community-level factors were significant in the multilevel analyses. Moving forward, it will be critical that health systems identify ways to address any barriers to care that populations might be experiencing, such as the use of mobile health services or telemedicine platforms. Additionally, public health emergency preparedness planning efforts must account for the unique needs of communities during future crises, to ensure that their health needs can continue to be met. Finally, additional research is needed to better understand how healthcare access and utilization practices have changed during versus before the pandemic., Competing Interests: S. Mehta receives material support from Abbott Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors do not have any other conflicts of interest to disclose, (Copyright: © 2024 Meyer 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.)

Objective: To assess the effect of World Trade Center (WTC) exposure on cardiovascular disease (CVD) in career firefighters. Methods: Firefighters from four US cities completed health questionnaires that provide information about demographics, CVD diagnoses, and CVD risk factors. Firefighters were also compared with respondents of the 2019 National Health Interview Survey. Results: Greater WTC exposure was positively associated with combined coronary artery disease, myocardial infarction, and angina (termed "CAD") when comparing WTC-exposed with non-WTC-exposed firefighters. Compared with the National Health Interview Survey population, firefighters had lower odds of CAD and stroke. Conclusions: An occupationally appropriate comparison is important to mitigate potential bias from the healthy worker effect. While the risk of CVD in WTC-exposed and non-WTC-exposed firefighters was significantly lower than a general US population, we observed an exposure gradient where greater WTC exposure was associated with greater odds of CVD., Competing Interests: Conflict of interest: H.C. received funding from the American Journal of Hypertension for work as a guest editor. All other authors have no conflicts of interest to declare., (Copyright © 2023 American College of Occupational and Environmental Medicine.)

Background: The degree to which routine, non-World Trade Center (WTC) firefighting exposures contribute to the WTC exposure-obstructive airway disease (OAD) relationship is unknown. Our objective was to compare the frequency of self-reported OAD diagnoses in WTC-exposed firefighters from the Fire Department of the City of New York (FDNY) compared with non-WTC-exposed firefighters from other cities and the general population., Methods: A total of 9792 WTC-exposed male FDNY firefighters and 3138 non-WTC-exposed male firefighters from Chicago, Philadelphia, and San Francisco who were actively employed on 9/11/01 and completed a health questionnaire were included. Logistic regression estimated odds ratios of self-reported asthma and COPD diagnoses in firefighters (WTC-exposed vs. non-WTC-exposed; all firefighters vs. general population), adjusting for age, race, smoking status, and last medical visit., Results: WTC-exposed firefighters were, on average, younger on 9/11 (mean ± SD = 40.2 ± 7.4 vs. 44.1 ± 9.1) and less likely to report ever-smoking (32.9% vs. 41.8%) than non-WTC-exposed firefighters. Odds of any OAD and asthma were 4.5 and 6.3 times greater, respectively, in WTC-exposed versus non-WTC-exposed. Odds of COPD were also greater in WTC-exposed versus non-WTC-exposed, particularly among never-smokers. Compared with the general population, WTC-exposed firefighters had greater odds of both asthma and COPD, while the nonexposed had lower odds of asthma and greater odds of COPD., Conclusions: Odds ratios for OAD diagnoses were greater in WTC-exposed firefighters versus both non-WTC-exposed and the general population after adjusting for covariates. While asthma and other OADs are known occupational hazards of firefighting, WTC exposure significantly compounded these adverse respiratory effects., (© 2023 The Authors. American Journal of Industrial Medicine published by Wiley Periodicals LLC.)

Background: Individuals with very low immunoglobulin E (IgE) levels have a high risk of developing malignancy. Previous studies have revealed that World Trade Center (WTC) responders exposed to carcinogens have an elevated risk of some cancers., Objective: To evaluate the association between low-serum IgE levels and cancer development in WTC-exposed responders., Methods: IgE levels were measured in 1851 WTC responders after September 11, 2001. This is the first pilot study in humans comparing the odds of developing cancer in this high-risk population, between the "low-IgE" (IgE in the lowest third percentile) vs "non-low-IgE" participants., Results: A significantly higher proportion of hematologic malignancies was found in low-IgE (4/55, 7.3%) compared with non-low-IgE (26/1796, 1.5%, P < .01) responders. The proportion of solid tumors were similar in both groups (5.5% vs 11.4%, P > .05). After adjustment for relevant confounders (race, sex, age at blood draw, WTC arrival time, smoking status), the low-IgE participants had 7.81 times greater odds (95% confidence interval, 1.77-29.35) of developing hematologic cancer when compared with non-low-IgE participants. The hematologic cancers found in this cohort were leukemia (n = 1), multiple myeloma (n = 1), and lymphoma (n = 2). No statistical significance was found when estimating the odds ratio for solid tumors in relation to IgE levels., Conclusion: WTC responders with low serum IgE levels had the highest odds of developing hematologic malignancies. This hypothesis-generating study suggests that low serum IgE levels might be associated with the development of specific malignancies in at-risk individuals exposed to carcinogens. Larger, multicenter studies with adequate follow-up of individuals with different IgE levels are needed to better evaluate this relationship., (Copyright © 2022 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

An elevated risk of myeloma precursor disease, monoclonal gammopathy of undetermined significance (MGUS), was identified among Fire Department of the City of New York (FDNY) World Trade Center (WTC)-exposed firefighters. Further investigation was needed to determine if these findings were reproducible in a more heterogeneous WTC-exposed rescue/recovery workers cohort, the Stony Brook University-General Responder Cohort GRC (SBU-GRC). MGUS risk was compared between the cohorts and to published general population estimates from Olmsted County, MN, USA. In this observational seroprevalence study, odds ratios (OR) and age-standardized risk ratios (RR) of MGUS (M-spike and light-chain-MGUS combined), M-spike, and light-chain-MGUS were estimated using logistic regression. Age-standardized prevalences were calculated for white males aged 50-79; RRs were estimated by comparing risk in the WTC-exposed cohort with the Olmsted County screened cohort. SBU-GRC had elevated odds of MGUS compared with FDNY (OR = 1.38; 95%CI = 1.00-1.89). The age-standardized prevalence of MGUS was 9.0/100 persons (95%CI = 7.5-10.6), over two-fold higher than the general population (RR = 2.08; 95%CI = 1.72-2.51); the age-standardized prevalence of light-chain-MGUS was 3.5-fold higher (RR = 3.54; 95%CI = 2.52-4.97). This study adds to mounting evidence supporting an association between WTC/environmental exposures and MGUS among rescue/recovery workers. Access to MGUS screenings for the entire WTC-exposed cohort could allow for treatment interventions that improve survival., (© 2022. The Author(s).)

Malaria incidence has declined in southern Zambia over recent decades, leading to efforts to achieve and sustain malaria elimination. Understanding the remaining disease burden is key to providing optimal health care. A longitudinal study conducted in a rural area of Choma District, Southern Province, Zambia, assessed the prevalence of and factors associated with symptoms of non-malarial illnesses and treatment-seeking behavior. We analyzed data collected monthly between October 2018 through September 2020 from 1,174 individuals from 189 households. No incident malaria cases were detected by rapid diagnostic tests among febrile participants. Mixed-effects logistic regression identified factors associated with cough, fever, diarrhea, and treatment-seeking. Incidence rates of cough (192 of 1,000 person-months), fever (87 of 1,000 person-months), and fever with cough (37 of 1,000 person-months) were highest among adults older than 65 years. Diarrhea incidence (37 of 1,000 person-months) was highest among children younger than 5 years. For every additional symptomatic household member, one's odds of experiencing symptoms increased: cough by 47% (95% CI, 40-55), fever by 31% (95% CI, 23-40), diarrhea by 31% (95% CI, 17-46), and fever with cough by 112% (95% CI, 90-137), consistent with household clustering of illnesses. However, between 35% and 75% of participants did not seek treatment for their symptoms. Treatment-seeking was most common for children 5 to 9 years old experiencing diarrhea (adjusted odds ratio, 3.61; 95% CI, 1.42-9.18). As malaria prevalence reduces, respiratory and diarrheal infections persist, particularly among young children but, notably, also among adults older than 65 years. Increasing awareness of the disease burden and treatment-seeking behavior are important for guiding resource re-allocation as malaria prevalence declines in this region.

Brain swelling occurs in cerebral malaria (CM) and may either reverse or result in fatal outcome. It is currently unknown how brain swelling in CM reverses, as brain swelling at the acute stage is difficult to study in humans and animal models with reliable induction of reversible edema are not known. In this study, we show that reversible brain swelling in experimental murine CM can be induced reliably after single vaccination with radiation-attenuated sporozoites as proven by in vivo high-field magnetic resonance imaging. Our results provide evidence that brain swelling results from transcellular blood-brain barrier disruption (BBBD), as revealed by electron microscopy. This mechanism enables reversal of brain swelling but does not prevent persistent focal brain damage, evidenced by microhemorrhages, in areas of most severe BBBD. In adult CM patients magnetic resonance imaging demonstrate microhemorrhages in more than one third of patients with reversible edema, emphasizing similarities of the experimental model and human disease. Our data suggest that targeting transcellular BBBD may represent a promising adjunct therapeutic approach to reduce edema and may improve neurological outcome., (© 2022 Jin et al.)

Background: Firefighting has been associated with posttraumatic stress disorder (PTSD) and other mental health conditions. We previously found that among Fire Department of the City of New York (FDNY) responders to the World Trade Center (WTC) disaster, higher-intensity WTC-exposure predicted PTSD symptoms, depressive symptoms, and subjective cognitive concerns. The present study aims to compare these symptoms in the FDNY WTC-exposed cohort versus a comparison cohort of non-FDNY, non-WTC-exposed firefighters., Methods: The study population included WTC-exposed male firefighters from FDNY (N = 8466) and non-WTC-exposed male firefighters from Chicago (N = 1195), Philadelphia (N = 770), and San Francisco (N = 650) fire departments who were employed on 9/11/2001 and completed a health questionnaire between 3/1/2018 and 12/31/2020. Current PTSD symptoms, depressive symptoms, and subjective cognitive concerns were assessed via validated screening instruments. Multivariable linear regression analyses stratified by fire department estimated the impact of covariates on each outcome., Results: Adjusted mean PTSD symptom scores ranged from 23.5 ± 0.6 in Chicago firefighters to 25.8 ± 0.2 in FDNY, and adjusted mean depressive symptom scores ranged from 7.3 ± 0.5 in Chicago to 9.4 ± 0.6 in Philadelphia. WTC-exposure was associated with fewer subjective cognitive concerns (β = -0.69 ± 0.05, p < .001) after controlling for covariates. Across cohorts, older age was associated with more cognitive concerns, but fewer PTSD and depressive symptoms., Conclusions: WTC-exposed firefighters had fewer cognitive concerns compared with non-WTC-exposed firefighters. We were unable to estimate associations between WTC exposure and PTSD symptoms or depressive symptoms due to variability between non-WTC-exposed cohorts. Longitudinal follow-up is needed to assess PTSD, depressive, and cognitive symptom trajectories in firefighter populations as they age., (© 2021 Wiley Periodicals LLC.)

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA ( PbA Ama1 OVA ) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1 -/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8 + T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1 -/- mice and wild type mice suffering from ECM. Importantly, CD8 + T cells were increased in the spleens of ECM-protected Ifnar1 -/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8 + T cell infiltration into the brain and increased ECM induction in PbA Ama1 OVA -infected Ifnar1 -/- mice. However, eosinophil-depletion did not reduce the CD8 + T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8 + T cell migration and proliferation during PbA Ama1 OVA -infection in Ifnar1 -/- mice and thereby are contributing to the protection from ECM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Scheunemann, Reichwald, Korir, Kuehlwein, Jenster, Hammerschmidt-Kamper, Lewis, Klocke, Borsche, Schwendt, Soun, Thiebes, Limmer, Engel, Mueller, Hoerauf, Hübner and Schumak.)

Mutations in presenilin 1 (PSEN1) or presenilin 2 (PSEN2), the catalytic subunit of γ-secretase, cause familial Alzheimer's disease (fAD). We hypothesized that mutations in PSEN1 reduce Notch signaling and alter neurogenesis. Expression data from developmental and adult neurogenesis show relative enrichment of Notch and γ-secretase expression in stem cells, whereas expression of APP and β-secretase is enriched in neurons. We observe premature neurogenesis in fAD iPSCs harboring PSEN1 mutations using two orthogonal systems: cortical differentiation in 2D and cerebral organoid generation in 3D. This is partly driven by reduced Notch signaling. We extend these studies to adult hippocampal neurogenesis in mutation-confirmed postmortem tissue. fAD cases show mutation-specific effects and a trend toward reduced abundance of newborn neurons, supporting a premature aging phenotype. Altogether, these results support altered neurogenesis as a result of fAD mutations and suggest that neural stem cell biology is affected in aging and disease., Competing Interests: Declaration of Interests H.Z. has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Pinteon Therapeutics, and CogRx; has given lectures in symposia sponsored by Fujirebio, Alzecure, and Biogen; and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. The other authors have no conflicts of interest to declare., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Clinical and experimental evidence suggests that the tuberculosis vaccine BCG offers protection against unrelated pathogens including the malaria parasite. Cerebral malaria (CM) is the most severe complication associated with Plasmodium falciparum infection in humans and is responsible for most of the fatalities attributed to malaria. We investigated whether BCG protected C57BL/6 mice from P. berghei ANKA (PbA)-induced experimental CM (ECM). The majority of PbA-infected mice that were immunized with BCG showed prolonged survival without developing clinical symptoms of ECM. However, this protective effect waned over time and was associated with the recovery of viable BCG from liver and spleen. Intriguingly, BCG-mediated protection from ECM was not associated with a reduction in parasite burden, indicating that BCG immunization did not improve anti-parasite effector mechanisms. Instead, we found a significant reduction in pro-inflammatory mediators and CD8 + T cells in brains of BCG-vaccinated mice. Together these data suggest that brain recruitment of immune cells involved in the pathogenesis of ECM decreased after BCG vaccination. Understanding the mechanisms underlying the protective effects of BCG on PbA-induced ECM can provide a rationale for developing effective adjunctive therapies to reduce the risk of death and brain damage in CM.

In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2-5 years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.

Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8 + T-cells and initiate immune responses against the parasites. Batf3 -/- mice lack a DC subset, which efficiently induces strong CD8 T-cell responses by cross-presentation of exogenous antigens. Here we show that Batf3 -/- mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood-brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3 -/- mice correlated with attenuated responses of cytotoxic T-cells, as their parasite-specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM-protected Batf3 -/- mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA-infected Batf3 -/- mice was associated with the absence of strong CD8 + T-cell activity and induction of immunoregulatory mediators and cells., (© 2019 The Authors. Immunology Published by John Wiley & Sons Ltd.)

The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ferlins mediate calcium-dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin-like protein (FLP) in host-to-vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent-mediated membrane lysis. In agreement with ferlin vesicular localisation, HA-tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin-mediated exocytosis., (© 2018 John Wiley & Sons Ltd.)

Objectives: To determine whether the Mini-Cog can be applied by nursing staff to hospitalized elderly patients for cognitive impairment associated risk stratification., Methods: This explorative prospective multicenter cohort study was carried out among 2522 patients aged 70 and older, hospitalized due to physical illness in eight hospitals in Rhineland-Palatinate, Germany. All patients were asked to conduct the Mini-Cog at the day of admission and were clustered into low-performance, intermediate-performance, and good-performance categories by trained nursing staff and two experienced geronto-psychiatrists as gold standard. Complications in the course of the treatment were monitored., Results: The Mini-Cog was conducted in 1398 (54%) out of 2522 eligible patients. Mini-Cog scores assessed by nursing staff differed from the gold standard in 327 cases (23.9%). According to the area under the curve (AUC), nursing staff identified cognitively low-performing patients almost as well as the geronto-psychiatrists (AUC = 0.862; 95% CI, 0.83-0.89; P < 0.001, accuracy 89.6%). Overall, 241 (17.6%) patients were classified as low performing. These patients had a significantly higher probability of suffering from at least one complication (odds ratio [OR] = 3.13; 95% CI, 2.09-4.70; calculated by a logistic regression model, adjusted for age), and they had a higher probability to show behavioral symptoms., Conclusion: Even under naturalistic conditions, nursing staff detected cognitively low-performing inpatients with the Mini-Cog. Using this short screening instrument should enable to predict complications of hospitalized older patients associated with cognitive impairment, a precondition to implement targeted care for this vulnerable patient group., (© 2018 John Wiley & Sons, Ltd.)

Purpose To investigate the association of inflammation and brain edema in a cerebral malaria (CM) mouse model with a combination of bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium, referred to as MPO-Gd, and cross-linked iron oxide nanoparticle (CLIO-NP) imaging. Materials and Methods Female wild-type (n = 23) and myeloperoxidase (MPO) knock-out (n = 5) mice were infected with the Plasmodium berghei ANKA strain from May 2016 to July 2018. Seven healthy mice served as control animals. At a Rapid Murine Coma and Behavioral Scale (RMCBS) score of less than 15, mice underwent MRI at 9.4 T and received gadodiamide, MPO-Gd, or CLIO-NPs. T1-weighted MRI was used to assess MPO activity, and T2*-weighted MRI was used to track CLIO-NPs. Immunofluorescent staining and flow cytometric analyses characterized CLIO-NPs, MPO, endothelial cells, and leukocytes. An unpaired, two-tailed Student t test was used to compare groups; Spearman correlation analysis was used to determine the relationship of imaging parameters to clinical severity. Results MPO-Gd enhancement occurred in inflammatory CM hotspots (olfactory bulb > rostral migratory stream > brainstem > cortex, P < .05 for all regions compared with control mice; mean olfactory bulb signal intensity ratio: 1.40 ± 0.07 vs 0.96 ± 0.01, P < .01). The enhancement was reduced in MPO knockout mice (mean signal intensity ratio at 60 minutes: 1.13 ± 0.04 vs 1.40 ± 0.07 in CM, P < .05). Blood-brain barrier compromise was suggested by parenchymal gadolinium enhancement, leukocyte recruitment, and endothelial activation. CLIO-NPs accumulated mainly intravascularly and at the vascular endothelium. CLIO-NPs were also found in the choroid plexus, indicating inflammation of the ventricular system. Blood-cerebrospinal fluid barrier breakdown showed correlation with brain swelling (r 2 : 0.55, P < .01) and RMCBS score (r 2 : 0.75, P < .001). Conclusion Iron oxide nanoparticle imaging showed strong inflammatory involvement of the microvasculature in a murine model of cerebral malaria. Furthermore, bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium imaging depicted parenchymal and intraventricular inflammation. This combined molecular imaging approach links vascular inflammation to breakdown of the blood-brain barrier and blood-cerebrospinal fluid barrier that correlate with global brain edema and disease severity. © RSNA, 2018 Online supplemental material is available for this article. See also the editorial by Kiessling in this issue.

Cerebral malaria is a complex neurological syndrome caused by an infection with Plasmodium falciparum parasites and is exclusively attributed to a series of host-parasite interactions at the pathological blood-stage of infection. In contrast, the preceding intra-hepatic phase of replication is generally considered clinically silent and thereby excluded from playing any role in the development of neurological symptoms. In this study, however, we present an antigen Pb maLS&#95;05 that is presented to the host immune system by both pre-erythrocytic and intra-erythrocytic stages and contributes to the development of cerebral malaria in mice. Although deletion of the endogenous Pb maLS&#95;05 prevented the development of experimental cerebral malaria (ECM) in susceptible mice after both sporozoite and infected red blood cell (iRBC) infections, we observed significant differences in contribution of the host immune response between both modes of inoculation. Moreover, Pb maLS&#95;05-specific CD8 + T cells contributed to the development of ECM after sporozoite but not iRBC-infection, suggesting that pre-erythrocytic antigens like Pb maLS&#95;05 can also contribute to the development of cerebral symptoms. Our data thus highlight the importance of the natural route of infection in the study of ECM, with potential implications for vaccine and therapeutic strategies against malaria.

Whole sporozoite vaccines represent one of the most promising strategies to induce protection against malaria. However, the development of efficient vaccination protocols still remains a major challenge. To understand how the generation of immunity is affected by variations in vaccination dosage and frequency, we systematically analyzed intrasplenic and intrahepatic CD8 + T cell responses following varied immunizations of mice with radiation-attenuated sporozoites. By combining experimental data and mathematical modeling, our analysis indicates a reversing role of spleen and liver in the generation of protective liver-resident CD8 + T cells during priming and booster injections: While the spleen acts as a critical source compartment during priming, the increase in vaccine-induced hepatic T cell levels is likely due to local reactivation in the liver in response to subsequent booster injections. Higher dosing accelerates the efficient generation of liver-resident CD8 + T cells by especially affecting their local reactivation. In addition, we determine the differentiation and migration pathway from splenic precursors toward hepatic memory cells thereby presenting a mechanistic framework for the impact of various vaccination protocols on these dynamics. Thus, our work provides important insights into organ-specific CD8 + T cell dynamics and their role and interplay in the formation of protective immunity against malaria.

The blood-stage of the Plasmodium parasite is one of the key phases within its life cycle that influences disease progression during a malaria infection. The efficiency of the parasite in infecting red blood cells (RBC) determines parasite load and parasite-induced hemolysis that is responsible for the development of anemia and potentially drives severe disease progression. However, the molecular factors defining the infectivity of Plasmodium parasites have not been completely identified so far. Using the Plasmodium berghei mouse model for malaria, we characterized and compared the blood-stage infection dynamics of Pb ANKA WT and a mutant parasite strain lacking a novel Plasmodium antigen, Pb maLS&#95;05, that is well conserved in both human and animal Plasmodium parasite strains. Infection of mice with parasites lacking PbmaLS&#95;05 leads to lower parasitemia levels and less severe disease progression in contrast to mice infected with the wildtype Pb ANKA strain. To specifically determine the effect of deleting PbmaLS&#95;05 on parasite infectivity we developed a mathematical model describing erythropoiesis and malarial infection of RBC. By applying our model to experimental data studying infection dynamics under normal and drug-induced altered erythropoietic conditions, we found that both Pb ANKA and PbmaLS&#95;05 (-) parasite strains differed in their infectivity potential during the early intra-erythrocytic stage of infection. Parasites lacking PbmaLS&#95;05 showed a decreased ability to infect RBC, and immature reticulocytes in particular that are usually a preferential target of the parasite. These altered infectivity characteristics limit parasite burden and affect disease progression. Our integrative analysis combining mathematical models and experimental data suggests that deletion of PbmaLS&#95;05 affects productive infection of reticulocytes, which makes this antigen a useful target to analyze the actual processes relating RBC preferences to the development of severe disease outcomes in malaria.

Cerebral malaria is a life-threatening complication of Plasmodia infection and a major cause of child mortality in Sub-Saharan Africa. We report that protection from experimental cerebral malaria in the rodent model is obtained by a single intravenous or subcutaneous whole-parasite immunization. Whole-parasite immunization with radiation-attenuated sporozoites was equally protective as immunization with non-attenuated sporozoites under chemoprophylaxis. Both immunization regimens delayed the development of blood-stage parasites, but differences in cellular and humoral immune mechanisms were observed. Single-dose whole-parasite vaccination might serve as a relatively simple and feasible immunization approach to prevent life-threatening cerebral malaria.

The intracellular development and differentiation of the Plasmodium parasite in the host liver is a prerequisite for the actual onset of malaria disease pathology. Since liver-stage infection is clinically silent and can be completely eliminated by sterilizing immune responses, it is a promising target for urgently needed innovative antimalarial drugs and/or vaccines. Discovered more than 65 years ago, these stages remain poorly understood regarding their molecular repertoire and interaction with their host cells in comparison to the pathogenic erythrocytic stages. The differentiating and replicative intrahepatic parasite resides in a membranous compartment called the parasitophorous vacuole, separating it from the host-cell cytoplasm. Here we outline seminal work that contributed to our present understanding of the fundamental dynamic cellular processes of the intrahepatic malarial parasite with both specific host-cell factors and compartments., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Plasmodium falciparum infections can cause severe malaria, but not every infected person develops life-threatening complications. In particular, carriers of the structural haemoglobinopathies S and C and infants are protected from severe disease. Protection is associated with impaired parasite-induced host actin reorganization, required for vesicular trafficking of parasite-encoded adhesins, and reduced cytoadherence of parasitized erythrocytes in the microvasculature. Here we show that aberrant host actin remodelling and the ensuing reduced cytoadherence result from a redox imbalance inherent to haemoglobinopathic and fetal erythrocytes. We further show that a transient oxidative insult to wild-type erythrocytes before infection with P. falciparum induces the phenotypic features associated with the protective trait of haemoglobinopathic and fetal erythrocytes. Moreover, pretreatment of mice with the pro-oxidative nutritional supplement menadione mitigate the development of experimental cerebral malaria. Our results identify redox imbalance as a causative principle of protection from severe malaria, which might inspire host-directed intervention strategies.

Long-term genetic data from intensively monitored natural populations are important for understanding how effective population sizes (Ne) can vary over time. We therefore genotyped 1622 common buzzard (Buteo buteo) chicks sampled over 12 consecutive years (2002-2013 inclusive) at 15 microsatellite loci. This data set allowed us to both compare single-sample with temporal approaches and explore temporal patterns in the effective number of parents that produced each cohort in relation to the observed population dynamics. We found reasonable consistency between linkage disequilibrium-based single-sample and temporal estimators, particularly during the latter half of the study, but no clear relationship between annual Ne estimates () and census sizes. We also documented a 14-fold increase in between 2008 and 2011, a period during which the census size doubled, probably reflecting a combination of higher adult survival and immigration from further afield. Our study thus reveals appreciable temporal heterogeneity in the effective population size of a natural vertebrate population, confirms the need for long-term studies and cautions against drawing conclusions from a single sample.

Background: Continuous pressure overload may lead to subclinical myocardial tissue changes in patients with hypertensive heart disease (HHD) and aortic stenosis (AS). The study aim was to detect interstitial fibrosis using quantitative cardiovascular magnetic resonance., Methods: Fifteen patients with HHD (arterial hypertension + septal wall thickness ≥13 mm), 33 with AS (eight mild, 15 moderate, 10 severe), and 60 healthy controls were enrolled. Native T1 maps (modified Look-Locker inversion recovery) were obtained in a basal, mid-ventricular, and apical shortaxis slice of the left ventricle to assess cardiac fibrosis. Focal fibrosis was assessed with late gadolinium enhancement (LGE)., Results: Patients with HHD and controls did not differ regarding the native myocardial T1 values, both per slice and per segment. In AS patients, apical native T1 values were lower than in controls, and there was a trend towards higher T1 values in the septum in severe AS (1172.6 ± 62.0 ms versus 1152.9 ± 43.9 ms). Five HHD patients and 11 AS patients had non-ischemic fibrosis in LGE images. Native T1 times did not differ between LGE-positive and LGEnegative groups (both with inclusion and exclusion of segments with LGE)., Conclusions: T1 mapping did not reveal any evidence of abnormal interstitial fibrosis in HHD subjects with mild hypertrophy. In severe AS, a trend towards more interstitial fibrosis was present, but absolute differences were small for decision making.

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.

The increasing use of screening technologies in malaria research has substantially expanded our knowledge on cellular factors hijacked by the Plasmodium parasite in the infected host, including those that participate in the clinically silent liver stage. This rapid gain in our understanding of the hepatic interaction partners now requires a means to validate and further disentangle parasite-host networks in physiologically relevant liver model systems. Here, we outline seminal work that contributed to our present knowledge on the intrahepatic Plasmodium host factors, followed by a discussion of surrogate models of mammalian livers or hepatocytes. We finally describe how Adeno-associated viruses could be engineered and used as hepatotropic tools to dissect Plasmodium-host interactions, and to deliberately control these networks for antimalaria vaccination or therapy., (© 2016 Federation of European Biochemical Societies.)

The biological function of the post-translational modification hypusine in the eukaryotic initiation factor 5A (EIF-5A) in eukaryotes is still not understood. Hypusine is formed by two sequential enzymatic steps at a specific lysine residue in the precursor protein EIF-5A. One important biological function of EIF-5A which was recently identified is the translation of polyproline-rich mRNA, suggesting its biological relevance in a variety of biological processes. Hypusinated eIF-5A controls the proliferation of cancer cells and inflammatory processes in malaria. It was shown that pharmacological inhibition of the enzymes involved in this pathway, deoxyhypusine synthase (DHS) and the deoxyhypusine hydroxylase (DOHH), arrested the growth of malaria parasites. Down-regulation of both the malarial eIF-5A and dhs genes by in vitro and in vivo silencing led to decreased transcript levels and protein expression and, in turn, to low parasitemia, confirming a critical role of hypusination in eIF-5A for proliferation in Plasmodium. To further investigate whether eIF-5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei. Full disruption of both genes was not successful; instead parasites harboring the episome for eIF-5A and dhs genes were obtained, suggesting that these genes may perform vital functions during the pathogenic blood cell stage. Next, a knock-in strategy was pursued for both endogenous genes eIF-5A and dhs from P. berghei. The latter resulted in viable recombinant parasites, strengthening the observation that they might be essential for proliferation during asexual development of the malaria parasite.

Resistance to radiation is considered to be an important reason for local failure after radiotherapy and tumor recurrence. However, the exact mechanisms of tumor resistance remain poorly understood. Current investigations of microRNAs as potential diagnostic and therapeutic tools for cancer treatment have shown promising results. With respect to radiotherapy resistance and response, there is now emerging evidence that microRNAs modulate key cellular pathways that mediate response to radiation. These data suggest that microRNAs might have significant potential as targets for the development of new therapeutic strategies to overcome radioresistance in cancer. This review summarizes the current literature pertinent to the influence of microRNAs in the response to radiotherapy for cancer treatment, with an emphasis on microRNAs as novel diagnostic and prognostic markers, as well as their potential to alter radiosensitivity.

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Intraguild predation (IGP) is a commonly recognized mechanism influencing the community structure of predators, but the complex interactions are notoriously difficult to disentangle. The mesopredator suppression hypothesis predicts that a superpredator may either simultaneously repress two mesopredators, restrain the dominant one and thereby release the subdominant mesopredator, or elicit different responses by both mesopredators. We show the outcome arising from such conditions in a three-level predator assemblage (Eurasian eagle owl Bubo bubo L., northern goshawk Accipiter gentilis L. and common buzzard Buteo buteo L.) studied over 25 years. In the second half of the study period, the eagle owl re-colonized the study area, thereby providing a natural experiment of superpredator introduction. We combined this set-up with detailed GIS analysis of habitat use and a field experiment simulating intrusion by the superpredator into territories of the subdominant mesopredator, the buzzard. Although population trends were positive for all three species in the assemblage, the proportion of failed breeding attempts increased significantly in both mesopredators after the superpredator re-colonized the area. We predicted that superpredator-induced niche shifts in the dominant mesopredator may facilitate mesopredator coexistence in superpredator-free refugia. We found significant changes in nesting habitat choice in goshawk, but not in buzzard. Since competition for enemy-free refugia and the rapid increase in population density may have constrained niche shifts of the subdominant mesopredator, we further predicted behavioural changes in response to the superpredator. The field experiment indeed showed a significant increase in aggressive response of buzzards towards eagle owl territory intrusion over the course of 10 years, probably due to phenotypic plasticity in the response towards superpredation risk. Overall, our results show that intraguild predation can be a powerful force of behavioural change, simultaneously influencing habitat use and aggressiveness in predator communities. These changes might help to buffer mesopredator populations against the negative effects of intraguild predation., (© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society.)

Background: Most patients with multiple sclerosis without previous optic neuritis have thinner retinal layers than healthy controls. We assessed the role of peripapillary retinal nerve fibre layer (pRNFL) thickness and macular volume in eyes with no history of optic neuritis as a biomarker of disability worsening in a cohort of patients with multiple sclerosis who had at least one eye without optic neuritis available., Methods: In this multicentre, cohort study, we collected data about patients (age ≥16 years old) with clinically isolated syndrome, relapsing-remitting multiple sclerosis, and progressive multiple sclerosis. Patients were recruited from centres in Spain, Italy, France, Germany, Czech Republic, Netherlands, Canada, and the USA, with the first cohort starting in 2008 and the latest cohort starting in 2013. We assessed disability worsening using the Expanded Disability Status Scale (EDSS). The pRNFL thickness and macular volume were assessed once at study entry (baseline) by optical coherence tomography (OCT) and was calculated as the mean value of both eyes without optic neuritis for patients without a history of optic neuritis or the value of the non-optic neuritis eye for patients with previous unilateral optic neuritis. Researchers who did the OCT at baseline were masked to EDSS results and the researchers assessing disability with EDSS were masked to OCT results. We estimated the association of pRNFL thickness or macular volume at baseline in eyes without optic neuritis with the risk of subsequent disability worsening by use of proportional hazards models that included OCT metrics and age, disease duration, disability, presence of previous unilateral optic neuritis, and use of disease-modifying therapies as covariates., Findings: 879 patients with clinically isolated syndrome (n=74), relapsing-remitting multiple sclerosis (n=664), or progressive multiple sclerosis (n=141) were included in the primary analyses. Disability worsening occurred in 252 (29%) of 879 patients with multiple sclerosis after a median follow-up of 2·0 years (range 0·5-5 years). Patients with a pRNFL of less than or equal to 87 μm or less than or equal to 88 μm (measured with Spectralis or Cirrus OCT devices) had double the risk of disability worsening at any time after the first and up to the third years of follow-up (hazard ratio 2·06, 95% CI 1·36-3·11; p=0·001), and the risk was increased by nearly four times after the third and up to the fifth years of follow-up (3·81, 1·63-8·91; p=0·002). We did not identify meaningful associations for macular volume., Interpretation: Our results provide evidence of the usefulness of monitoring pRNFL thickness by OCT for prediction of the risk of disability worsening with time in patients with multiple sclerosis., Funding: Instituto de Salud Carlos III., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

It is poorly understood how progressive brain swelling in experimental cerebral malaria (ECM) evolves in space and over time, and whether mechanisms of inflammation or microvascular sequestration/obstruction dominate the underlying pathophysiology. We therefore monitored in the Plasmodium berghei ANKA-C57BL/6 murine ECM model, disease manifestation and progression clinically, assessed by the Rapid-Murine-Coma-and-Behavioral-Scale (RMCBS), and by high-resolution in vivo MRI, including sensitive assessment of early blood-brain-barrier-disruption (BBBD), brain edema and microvascular pathology. For histological correlation HE and immunohistochemical staining for microglia and neuroblasts were obtained. Our results demonstrate that BBBD and edema initiated in the olfactory bulb (OB) and spread along the rostral-migratory-stream (RMS) to the subventricular zone of the lateral ventricles, the dorsal-migratory-stream (DMS), and finally to the external capsule (EC) and brainstem (BS). Before clinical symptoms (mean RMCBS = 18.5±1) became evident, a slight, non-significant increase of quantitative T2 and ADC values was observed in OB+RMS. With clinical manifestation (mean RMCBS = 14.2±0.4), T2 and ADC values significantly increased along the OB+RMS (p = 0.049/p = 0.01). Severe ECM (mean RMCBS = 5±2.9) was defined by further spread into more posterior and deeper brain structures until reaching the BS (significant T2 elevation in DMS+EC+BS (p = 0.034)). Quantitative automated histological analyses confirmed microglial activation in areas of BBBD and edema. Activated microglia were closely associated with the RMS and neuroblasts within the RMS were severely misaligned with respect to their physiological linear migration pattern. Microvascular pathology and ischemic brain injury occurred only secondarily, after vasogenic edema formation and were both associated less with clinical severity and the temporal course of ECM. Altogether, we identified a distinct spatiotemporal pattern of microglial activation in ECM involving primarily the OB+RMS axis, a distinct pathway utilized by neuroblasts and immune cells. Our data suggest significant crosstalk between these two cell populations to be operative in deeper brain infiltration and further imply that the manifestation and progression of cerebral malaria may depend on brain areas otherwise serving neurogenesis.

Background: Genome editing of malaria parasites is key to the generation of live attenuated parasites used in experimental vaccination approaches. DNA repair in Plasmodium generally occurs only through homologous recombination. This has been used to generate transgenic parasites that lack one to three genes, leading to developmental arrest in the liver and allowing the host to launch a protective immune response. While effective in principle, this approach is not safe for use in humans as single surviving parasites can still cause disease. Here we use zinc-finger nucleases to generate attenuated parasite lines lacking an entire chromosome arm, by a timed induction of a double-strand break. Rare surviving parasites also allow the investigation of unconventional DNA repair mechanisms in a rodent malaria parasite., Results: A single, zinc-finger nuclease-induced DNA double-strand break results in the generation of attenuated parasite lines that show varying degrees of developmental arrest, protection efficacy in an immunisation regime and safety, depending on the timing of zinc-finger nuclease expression within the life cycle. We also identify DNA repair by microhomology-mediated end joining with as little as four base pairs, resulting in surviving parasites and thus breakthrough infections., Conclusions: Malaria parasites can repair DNA double-strand breaks with surprisingly small mini-homology domains located across the break point. Timely expression of zinc-finger nucleases could be used to generate a new generation of attenuated parasite lines lacking hundreds of genes.

Parents may adapt their offspring sex ratio in response to their own phenotype and environmental conditions. The most significant causes for adaptive sex-ratio variation might express themselves as different distributions of fitness components between sexes along a given variable. Several causes for differential sex allocation in raptors with reversed sexual size dimorphism have been suggested. We search for correlates of fledgling sex in an extensive dataset on common buzzards Buteo buteo, a long-lived bird of prey. Larger female offspring could be more resource-demanding and starvation-prone and thus the costly sex. Prominent factors such as brood size and laying date did not predict nestling sex. Nonetheless, lifetime sex ratio (LSR, potentially indicative of individual sex allocation constraints) and overall nestling sex were explained by territory quality with more females being produced in better territories. Additionally, parental plumage morphs and the interaction of morph and prey abundance tended to explain LSR and nestling sex, indicating local adaptation of sex allocation However, in a limited census of nestling mortality, not females but males tended to die more frequently in prey-rich years. Also, although females could have potentially longer reproductive careers, a subset of our data encompassing full individual life histories showed that longevity and lifetime reproductive success were similarly distributed between the sexes. Thus, a basis for adaptive sex allocation in this population remains elusive. Overall, in common buzzards most major determinants of reproductive success appeared to have no effect on sex ratio but sex allocation may be adapted to local conditions in morph-specific patterns.

Cerebral malaria is one of the most severe complications of malaria disease, attributed to a complicated series of immune reactions in the host. The syndrome is marked by inflammatory immune responses, margination of leukocytes, and parasitized erythrocytes in cerebral vessels leading to breakdown of the blood-brain barrier. We show that chemical attenuation of the parasite at the very early, clinically silent liver stage suppresses parasite development, delays the time until parasites establish blood-stage infection, and provokes an altered host immune response, modifying immunopathogenesis and protecting from cerebral disease. The early response is proinflammatory and cell mediated, with increased T cell activation in the liver and spleen, and greater numbers of effector T cells, cytokine-secreting T cells, and proliferating, proinflammatory cytokine-producing T cells. Dendritic cell numbers, T cell activation, and infiltration of CD8(+) T cells to the brain are decreased later in infection, possibly mediated by the anti-inflammatory cytokine IL-10. Strikingly, protection can be transferred to naive animals by adoptive transfer of lymphocytes from the spleen at very early times of infection. Our data suggest that a subpopulation belonging to CD8(+) T cells as early as day 2 postinfection is responsible for protection. These data indicate that liver stage-directed early immune responses can moderate the overall downstream host immune response and modulate severe malaria outcome., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Sclerosing angiomatoid nodular transformation of the spleen (SANT) is a benign, extremely rare vascular lesion of the spleen with unknown pathogenesis. SANT is often discovered incidentally, and can sometimes be found in patients with a history of cancer. Based on absent definitive radiological signs and varying growth patterns, distinction from malignant processes such as metastasis can be very difficult. Therefore, surgical resection of the spleen is indicated in most cases of patients with history of cancer. We report a case of a bifocal manifestation of SANT in the spleen in a patient with history of colon cancer and newly-diagnosed metachronous liver metastases.

Background: Whole-parasite immunization remains the benchmark in malaria vaccine development. A major bottleneck in the translation of whole-parasite immunization towards routine vaccination is the mode of administration, since high degrees of protection are currently only achieved by intravenous, and not by intradermal or subcutaneous injection of viable parasites. It is known that only a small proportion of subcutaneously administered parasites reach the subsequent liver stage and low parasite liver load was shown to be associated with low protective efficacy. The objective of this analysis was to evaluate whether the liver load following subcutaneous parasite injection could be augmented by co-administration of pro-inflammatory or anti-coagulatory drugs., Methods: In the C57BL/6 Plasmodium berghei ANKA model, the clinical outcome (time to patent blood stage infection and survival) and relative parasite liver load was assessed in mice infected by subcutaneous or intramuscular sporozoite (SPZ) administration in the presence or absence of histamine and heparin supplementation in comparison to intravenously administered SPZ. In addition, a vaccination experiment was carried out to assess the protective efficacy of an improved, histamine-supplemented subcutaneous immunization regimen., Results: The parasite liver load following subcutaneous SPZ administration can be significantly increased by co-administration of histamine and heparin. A dose-dependent relation between parasite liver load and histamine dosage was observed. However, despite a relatively high parasite liver load, the protective efficacy of histamine-supplemented subcutaneous immunization remains inferior as compared to intravenous SPZ administration., Conclusions: Histamine supplementation might facilitate the future development of a non-intravenous whole-parasite vaccine. Further investigations are needed to reveal the effect of histamine supplementation and subcutaneous SPZ administration on the acquisition of protective immunity.

wALADin1 benzimidazoles are specific inhibitors of δ-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 μM, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sterile attenuation of Plasmodium parasites at the liver-stage either by irradiation or genetic modification, or at the blood-stage by chemoprophylaxis, has been shown to induce immune responses that can protect against subsequent wild-type infection. However, following certain interventions, parasite attenuation can be incomplete or non-sterile. Instead parasites are rendered developmentally stunted but still capable of establishing an acute infection. In experiments involving Plasmodium berghei ANKA, a model of experimental cerebral malaria, it has been observed that several forms of attenuated parasites do not induce cerebral pathology. In this perspective we collect evidence from studies on murine malaria in particular, and attempt to "connect the dots" between early immune responses and protection from severe cerebral disease, highlighting potential parallels to human infection.

Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)