Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

Widespread breakdown in masting in European beech due to rising summer temperatures.

Climate change effects on tree reproduction are poorly understood, even though the resilience of populations relies on sufficient regeneration to balance increasing rates of mortality. Forest-forming tree species often mast, i.e. reproduce through synchronised year-to-year variation in seed production, which improves pollination and reduces seed predation. Recent observations in European beech show, however, that current climate change can dampen interannual variation and synchrony of seed production and that this masting breakdown drastically reduces the viability of seed crops. Importantly, it is unclear under which conditions masting breakdown occurs and how widespread breakdown is in this pan-European species. Here, we analysed 50 long-term datasets of population-level seed production, sampled across the distribution of European beech, and identified increasing summer temperatures as the general driver of masting breakdown. Specifically, increases in site-specific mean maximum temperatures during June and July were observed across most of the species range, while the interannual variability of population-level seed production (CVp) decreased. The declines in CVp were greatest, where temperatures increased most rapidly. Additionally, the occurrence of crop failures and low seed years has decreased during the last four decades, signalling altered starvation effects of masting on seed predators. Notably, CVp did not vary among sites according to site mean summer temperature. Instead, masting breakdown occurs in response to warming local temperatures (i.e. increasing relative temperatures), such that the risk is not restricted to populations growing in warm average conditions. As lowered CVp can reduce viable seed production despite the overall increase in seed count, our results warn that a covert mechanism is underway that may hinder the regeneration potential of European beech under climate change, with great potential to alter forest functioning and community dynamics.

Author Correction: Environment and host as large-scale controls of ectomycorrhizal fungi.

Change history: In the HTML version of this Article, author 'Filipa Cox' had no affiliation in the author list, although she was correctly associated with affiliation 3 in the PDF. In addition, the blue circles for 'oak' were missing from Extended Data Fig. 1. These errors have been corrected online.

Environment and host as large-scale controls of ectomycorrhizal fungi.

Explaining the large-scale diversity of soil organisms that drive biogeochemical processes-and their responses to environmental change-is critical. However, identifying consistent drivers of belowground diversity and abundance for some soil organisms at large spatial scales remains problematic. Here we investigate a major guild, the ectomycorrhizal fungi, across European forests at a spatial scale and resolution that is-to our knowledge-unprecedented, to explore key biotic and abiotic predictors of ectomycorrhizal diversity and to identify dominant responses and thresholds for change across complex environmental gradients. We show the effect of 38 host, environment, climate and geographical variables on ectomycorrhizal diversity, and define thresholds of community change for key variables. We quantify host specificity and reveal plasticity in functional traits involved in soil foraging across gradients. We conclude that environmental and host factors explain most of the variation in ectomycorrhizal diversity, that the environmental thresholds used as major ecosystem assessment tools need adjustment and that the importance of belowground specificity and plasticity has previously been underappreciated.

Neurotrophic Factors as Regenerative Therapy for Neurodegenerative Diseases: Current Status, Challenges and Future Perspectives.

Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), spinal cord injury (SCI), and amyotrophic lateral sclerosis (ALS), are characterized by acute or chronic progressive loss of one or several neuronal subtypes. However, despite their increasing prevalence, little progress has been made in successfully treating these diseases. Research has recently focused on neurotrophic factors (NTFs) as potential regenerative therapy for neurodegenerative diseases. Here, we discuss the current state of knowledge, challenges, and future perspectives of NTFs with a direct regenerative effect in chronic inflammatory and degenerative disorders. Various systems for delivery of NTFs, such as stem and immune cells, viral vectors, and biomaterials, have been applied to deliver exogenous NTFs to the central nervous system, with promising results. The challenges that currently need to be overcome include the amount of NTFs delivered, the invasiveness of the delivery route, the blood-brain barrier permeability, and the occurrence of side effects. Nevertheless, it is important to continue research and develop standards for clinical applications. In addition to the use of single NTFs, the complexity of chronic inflammatory and degenerative diseases may require combination therapies targeting multiple pathways or other possibilities using smaller molecules, such as NTF mimetics, for effective treatment.

Phase 1 Randomized, Placebo-Controlled, Dose-Escalating Study to Evaluate OVX836, a Nucleoprotein-Based Influenza Vaccine: Intramuscular Results.

BACKGROUND: OVX836 is a recombinant protein vaccine targeting the highly conserved influenza nucleoprotein (NP), which could confer broad-spectrum protection against this disease. METHODS: A randomized, placebo-controlled, double-blind, dose-escalating, single- center, first-in-human study was conducted in 36 healthy adults aged 18-49 years. Twelve subjects per cohort (9 vaccine and 3 placebo) received 2 OVX836 intramuscular administrations on days 1 and 28 at the dose level of 30 µg, 90 µg, or 180 µg. Safety and immunogenicity were assessed after each vaccination and for 150 days in total. RESULTS: OVX836 was safe and well tolerated at all dose levels, with no difference in solicited local and systemic symptoms, and unsolicited adverse events between the first and second administration, or between dose levels. All subjects presented pre-existing NP-specific immunity at baseline. OVX836 induced a significant increase in NP-specific interferon-gamma T cells and anti-NP immunoglobulin G at all dose levels after the first vaccination. The second vaccination did not further increase the response. There was a trend for a dose effect in the immune response. CONCLUSIONS: The safety and reactogenicity profile, as well as the humoral and cellular immune responses, encourage further evaluation of OVX836 in a larger Phase 2a study.

Impact of Maternal Pertussis Antibodies on the Infants' Cellular Immune Responses.

INTRODUCTION: Maternal antibody interference of the infant's humoral immune responses raises some concern to the strategy of maternal Tdap (tetanus, diphtheria, acellular pertussis [aP]) vaccination. This study assessed the impact of maternal Tdap antibodies on the infant's pertussis-specific T lymphocyte responses following infant vaccination with an aP containing vaccine, in a term and preterm born cohort. METHODS: Heparin samples (±0.5 mL) were conveniently drawn from infants of a Belgian prospective cohort study (N = 79, NCT02511327), including Tdap vaccinated (Boostrix®) and nonvaccinated women (no Tdap vaccine in the last 5 years) that delivered at term or prematurely. Sampling was performed before and 1 month after primary (8-12-16 weeks) and booster vaccination (13 or 15 months) with DTaP-IPV-HB-PRP~T vaccine (Hexyon®). Pertussis toxin (PT)-specific CD3+, CD3+ CD4+ and CD3+ CD8+ lymphoblasts and their cytokine secretions were measured using a flow cytometric assay on whole blood (FASCIA) and multiplex technology (Meso Scale Discovery), respectively. RESULTS: In total, 57% of all infants were considered PT-specific CD3+ CD4+ lymphoblasts responders after primary and booster vaccination, whereas 17% were CD3+ CD8+ lymphoblast responders. Interferon (IFN)-γ, interleukin (IL)-13, IL-17A, and IL-5 cytokine secretions after primary and booster vaccination were indicative of a mixed T helper (Th) 1/Th2/Th17 cell profile. Lymphoblast and cytokine levels were comparable between term and preterm infants. Nonresponders for IL-13 after booster vaccination had higher maternal PT immunoglobulin G (IgG) levels at birth when compared to responders. CONCLUSIONS: Term and preterm born infants are capable of inducing Th1, Th2, and Th17 responses after aP vaccination, yet maternal vaccination modulate these responses. Evaluation of this effect in larger trials is needed.

Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics.

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner.

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4+CD127-CD25hi Tregs and CD4+CD127-CD25+CD45RA+ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.

Tolerogenic Dendritic Cells Induce Apoptosis-Independent T Cell Hyporesponsiveness of SARS-CoV-2-Specific T Cells in an Antigen-Specific Manner.

Although the global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, there are currently no specific and highly efficient drugs for COVID-19 available, particularly in severe cases. Recent findings demonstrate that severe COVID-19 disease that requires hospitalization is associated with the hyperactivation of CD4+ and CD8+ T cell subsets. In this study, we aimed to counteract this high inflammatory state by inducing T-cell hyporesponsiveness in a SARS-CoV-2-specific manner using tolerogenic dendritic cells (tolDC). In vitro-activated SARS-CoV-2-specific T cells were isolated and stimulated with SARS-CoV-2 peptide-loaded monocyte-derived tolDC or with SARS-CoV-2 peptide-loaded conventional (conv) DC. We demonstrate a significant decrease in the number of interferon (IFN)-γ spot-forming cells when SARS-CoV-2-specific T cells were stimulated with tolDC as compared to stimulation with convDC. Importantly, this IFN-γ downmodulation in SARS-CoV-2-specific T cells was antigen-specific, since T cells retain their capacity to respond to an unrelated antigen and are not mediated by T cell deletion. Altogether, we have demonstrated that SARS-CoV-2 peptide-pulsed tolDC induces SARS-CoV-2-specific T cell hyporesponsiveness in an antigen-specific manner as compared to stimulation with SARS-CoV-2-specific convDC. These observations underline the clinical potential of tolDC to correct the immunological imbalance in the critically ill.

Made to Measure: Patient-Tailored Treatment of Multiple Sclerosis Using Cell-Based Therapies.

Currently, there is still no cure for multiple sclerosis (MS), which is an autoimmune and neurodegenerative disease of the central nervous system. Treatment options predominantly consist of drugs that affect adaptive immunity and lead to a reduction of the inflammatory disease activity. A broad range of possible cell-based therapeutic options are being explored in the treatment of autoimmune diseases, including MS. This review aims to provide an overview of recent and future advances in the development of cell-based treatment options for the induction of tolerance in MS. Here, we will focus on haematopoietic stem cells, mesenchymal stromal cells, regulatory T cells and dendritic cells. We will also focus on less familiar cell types that are used in cell therapy, including B cells, natural killer cells and peripheral blood mononuclear cells. We will address key issues regarding the depicted therapies and highlight the major challenges that lie ahead to successfully reverse autoimmune diseases, such as MS, while minimising the side effects. Although cell-based therapies are well known and used in the treatment of several cancers, cell-based treatment options hold promise for the future treatment of autoimmune diseases in general, and MS in particular.

Cells to the Rescue: Emerging Cell-Based Treatment Approaches for NMOSD and MOGAD.

Cell-based therapies are gaining momentum as promising treatments for rare neurological autoimmune diseases, including neuromyelitis optica spectrum disorders and myelin oligodendrocyte glycoprotein antibody-associated disease. The development of targeted cell therapies is hampered by the lack of adequate animal models that mirror the human disease. Most cell-based treatments, including HSCT, CAR-T cell, tolerogenic dendritic cell and mesenchymal stem cell treatment have entered early stage clinical trials or have been used as rescue treatment in treatment-refractory cases. The development of antigen-specific cell-based immunotherapies for autoimmune diseases is slowed down by the rarity of the diseases, the lack of surrogate outcomes and biomarkers that are able to predict long-term outcomes and/or therapy effectiveness as well as challenges in the manufacturing of cellular products. These challenges are likely to be overcome by future research.

Regulating the regulators: Is introduction of an antigen-specific approach in regulatory T cells the next step to treat autoimmunity?

In autoimmunity, the important and fragile balance between immunity and tolerance is disturbed, resulting in abnormal immune responses to the body's own tissues and cells. CD4+CD25hiFoxP3+ regulatory T cells (Tregs) induce peripheral tolerance in vivo by means of direct cell-cell contact and release of soluble factors, or indirectly through antigen-presenting cells (APC), thereby controlling auto-reactive effector T cells. Based on these unique capacities of Tregs, preclinical studies delivered proof-of-principle for the clinical use of Tregs for the treatment of autoimmune diseases. To date, the first clinical trials using ex vivo expanded polyclonal Tregs have been completed. These pioneering studies demonstrate the feasibility of generating large numbers of polyclonal Tregs in a good manufacturing practices (GMP)-compliant manner, and that infusion of Tregs is well tolerated by patients with no evidence of general immunosuppression. Nonetheless, only modest clinical results were observed, arguing that a more antigen-specific approach might be needed to foster a durable patient-specific clinical cell therapy without the risk for general immunosuppression. In this review, we discuss current knowledge, applications and future goals of adoptive immune-modulatory Treg therapy for the treatment of autoimmune disease and transplant rejection. We describe the key advances and prospects of the potential use of T cell receptor (TCR)- and chimeric antigen receptor (CAR)-engineered Tregs in future clinical applications. These approaches could deliver the long-awaited breakthrough in stopping undesired autoimmune responses and transplant rejections.

Towards comparable assessment of the soil nutrient status across scales-Review and development of nutrient metrics.

Nutrient availability influences virtually every aspect of an ecosystem, and is a critical modifier of ecosystem responses to global change. Although this crucial role of nutrient availability in regulating ecosystem structure and functioning has been widely acknowledged, nutrients are still often neglected in observational and experimental synthesis studies due to difficulties in comparing the nutrient status across sites. In the current study, we explain different nutrient-related concepts and discuss the potential of soil-, plant- and remote sensing-based metrics to compare the nutrient status across space. Based on our review and additional analyses on a dataset of European, managed temperate and boreal forests (ICP [International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests] Forests dataset), we conclude that the use of plant- and remote sensing-based metrics that rely on tissue stoichiometry is limited due to their strong dependence on species identity. The potential use of other plant-based metrics such as Ellenberg indicator values and plant-functional traits is also discussed. We conclude from our analyses and review that soil-based metrics have the highest potential for successful intersite comparison of the nutrient status. As an example, we used and adjusted a soil-based metric, previously developed for conifer forests across Sweden, against the same ICP Forests data. We suggest that this adjusted and further adaptable metric, which included the organic carbon concentration in the upper 20 cm of the soil (including the organic fermentation-humus [FH] layer), the C:N ratio and pHCaCl2 of the FH layer, can be used as a complementary tool along with other indicators of nutrient availability, to compare the background nutrient status across temperate and boreal forests dominated by spruce, pine or beech. Future collection and provision of harmonized soil data from observational and experimental sites is crucial for further testing and adjusting the metric.

FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations.

Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia.

Relapse is a major problem in acute myeloid leukemia (AML) and adversely affects survival. In this phase 2 study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms' tumor 1 (WT1) messenger RNA (mRNA) as postremission treatment in 30 patients with AML at very high risk of relapse. There was a demonstrable antileukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which were sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in nonresponders (53.8% vs 25.0%; P = .01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25%, and 5-year relapse-free survival was higher in responders than in nonresponders (50% vs 7.7%; P < .0001). In patients age ≤65 and >65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared with 51.7% and 18% in the Swedish Acute Leukemia Registry. Long-term clinical response was correlated with increased circulating frequencies of polyepitope WT1-specific CD8+ T cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed-type hypersensitivity-infiltrating CD8+ T lymphocytes. In conclusion, vaccination of patients with AML with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224.

The response of soil solution chemistry in European forests to decreasing acid deposition.

Acid deposition arising from sulphur (S) and nitrogen (N) emissions from fossil fuel combustion and agriculture has contributed to the acidification of terrestrial ecosystems in many regions globally. However, in Europe and North America, S deposition has greatly decreased in recent decades due to emissions controls. In this study, we assessed the response of soil solution chemistry in mineral horizons of European forests to these changes. Trends in pH, acid neutralizing capacity (ANC), major ions, total aluminium (Altot ) and dissolved organic carbon were determined for the period 1995-2012. Plots with at least 10 years of observations from the ICP Forests monitoring network were used. Trends were assessed for the upper mineral soil (10-20 cm, 104 plots) and subsoil (40-80 cm, 162 plots). There was a large decrease in the concentration of sulphate (SO42-) in soil solution; over a 10-year period (2000-2010), SO42- decreased by 52% at 10-20 cm and 40% at 40-80 cm. Nitrate was unchanged at 10-20 cm but decreased at 40-80 cm. The decrease in acid anions was accompanied by a large and significant decrease in the concentration of the nutrient base cations: calcium, magnesium and potassium (Bc = Ca2+  + Mg2+  + K+ ) and Altot over the entire dataset. The response of soil solution acidity was nonuniform. At 10-20 cm, ANC increased in acid-sensitive soils (base saturation ≤10%) indicating a recovery, but ANC decreased in soils with base saturation >10%. At 40-80 cm, ANC remained unchanged in acid-sensitive soils (base saturation ≤20%, pHCaCl2 ≤ 4.5) and decreased in better-buffered soils (base saturation >20%, pHCaCl2 > 4.5). In addition, the molar ratio of Bc to Altot either did not change or decreased. The results suggest a long-time lag between emission abatement and changes in soil solution acidity and underline the importance of long-term monitoring in evaluating ecosystem response to decreases in deposition.

In situ proximity of CX3CR1-positive mononuclear phagocytes and VIP-ergic nerve fibers suggests VIP-ergic immunomodulation in the mouse ileum.

Being continuously exposed to a plethora of antigens ranging from food antigens to potential pathogenic organisms, the gastrointestinal (GI) tract harbors the largest collection of immune cells in the mammalian body. This immune system has to maintain a delicate balance between mounting an active immune response and maintaining tolerance. The GI tract is also home to an elaborate intrinsic nervous system, the enteric nervous system (ENS). Various in vitro studies of neuro-immune communication have suggested that vasoactive intestinal peptide (VIP), an important GI neurotransmitter, modulates mononuclear phagocytes (MNPs), i.e., dendritic cells and macrophages. Using a combined approach of reverse transcription plus the polymerase chain reaction, immunofluorescence, three-dimensional maximum intensity projections and immunoelectron microscopy, we investigate the interaction between the enteric innervation and MNPs in the ileal lamina propria (LP). We demonstrate that VIP-ergic fibers of the ENS lie adjacent to CX3CR1+ MNPs and that VPAC1 is constitutively expressed on ileal CX3CR1+ cells in the LP of the mouse. We also identify, for the first time, CX3CR1+ immune cells in the LP at the ultrastructural level. Our data thus reveal the in situ presence of the molecular components that are necessary for a VIP-mediated neuro-immune interaction between the ENS and CX3CR1-expressing immune cells in the LP of the ileum.

Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood-Brain Barrier under Inflammatory Conditions In Vitro.

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood-brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.