Response Patterns Of Cells Research Articles

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Combining sandblasting and pink anodisation of Ti implants as a promising method for improving fibroblast adhesion and immune response.

This study examined the effect of combining the sandblasting and anodising of titanium alloys used in implants on the cell response and protein adsorption patterns. The titanium samples were divided into four groups depending on the surface treatment: machining (MC), pink anodisation (PA), sandblasting (MC04) and a combination of the last two (MC04 + PA). Their physicochemical properties were analysed by SEM/EDX, Raman, contact angle measurements and profilometry. In vitro responses were examined using human gingival fibroblastic (HGF) cells and THP-1 macrophages. Cytokine secretion, macrophage adhesion and gene expression were measured by ELISA, confocal microscopy and RT-PCR. Cell adhesion and collagen secretion were evaluated in HGF cultures. The adsorption of immune and regenerative proteins onto the surfaces was assessed employing nLC-MS/MS. MC04 + PA surfaces exhibited a change in the roughness, chemical composition and hydrophilicity of the material, showing more elongated HGF cells and a considerable increase in the area of cells exposed to the MC04 + PA surfaces. Moreover, cells cultured on MC04 + PA generally showed a reduction in the expression of proinflammatory genes (TNF-α, MCP-1, C5, NF-kB and ICAM-1) and an increase in the secretion of anti-inflammatory cytokines, such as IL-4. These results correlated with the proteomic data; we found preferential adsorption of proteins favouring cell adhesion, such as DSC1 and PCOC1. A considerable reduction in the adsorption of immunoglobulins and proteins associated with acute inflammatory response (including SAA4) was also observed. The study highlights the potential advantages of MC04 + PA surface treatment to modify dental implant abutments; it enhances their compatibility with soft tissues and reduces the inflammatory response.

MRNA sequencing and CyTOF analysis revealed ASPP2 altered the response patterns of hepatocellular carcinoma HepG2 cells to usnic acid.

In a previous study, our team found that ASPP2 overexpression increases the sensitivity of liver cancer cells to sorafenib. ASPP2 is an important target in the study of drug therapy for hepatocellular carcinoma. In this study, we demonstrated that ASPP2 altered the response of HepG2 cells to usnic acid (UA) by using mRNA sequencing and CyTOF. CCK8 assay was used to detect cytotoxicity of UA on HepG2 cells. Annexin V-RPE assay, TUNEL assay, and cleaved caspase 3 assay were performed to examine the apoptotic cell death induced by UA. Transcriptomic sequencing and a single-cell mass cytometry were used to analyze the dynamic response of HepG2shcon and HepG2shASPP2 cells to UA treatment. We have demonstrated that UA could inhibit proliferation in HepG2 cells in a concentration-dependent manner. Apoptotic cell death was significantly induced by UA in HepG2 cells, while knocking down ASPP2 could increase the resistance of HepG2 cells to UA. Data from mRNA-Seq indicated that knockout of ASPP2 in HepG2 cells affected cell proliferation, cycle, and metabolism. ASPP2 knockdown resulted in increased stemness and decreased apoptosis of HepG2 cells under the action of UA. CyTOF analysis confirmed the above results, ASPP2 knockdown increased oncoproteins in HepG2 cells and altered response patterns of HepG2 cells to UA. Our data suggested that the natural compound UA could inhibit liver cancer HepG2 cells; meanwhile, ASPP2 knockdown could affect response patterns of HepG2 cells to UA. The above results indicate that ASPP2 could be a research target in the chemoresistance of liver cancer.

Single-cell transcriptomics reveals distinct cell response between acute and chronic pulmonary infection of Pseudomonas aeruginosa.

Knowledge of the changes in the immune microenvironment during pulmonary bacterial acute and chronic infections is limited. The dissection of immune system may provide a basis for effective therapeutic strategies against bacterial infection. Here, we describe a single immune cell atlas of mouse lungs after acute and chronic Pseudomonas aeruginosa infection using single-cell transcriptomics, multiplex immunohistochemistry, and flow cytometry. Our single-cell transcriptomic analysis revealed large-scale comprehensive changes in immune cell composition and high variation in cell-cell interactions after acute and chronic P. aeruginosa infection. Bacterial infection reprograms the genetic architecture of immune cell populations. We identified specific immune cell types, including Cxcl2+ B cells and interstitial macrophages, in response to acute and chronic infection, such as their proportions, distribution, and functional status. Importantly, the patterns of immune cell response are drastically different between acute and chronic infection models. The distinct molecular signatures highlight the importance of the highly dynamic cell-cell interaction process in different pathological conditions, which has not been completely revealed previously. These findings provide a comprehensive and unbiased immune cellular landscape for respiratory pathogenesis in mice, enabling further understanding of immunologic mechanisms in infection and inflammatory diseases.

Metabolic requirements of Th17 cells and of B cells: Regulation and defects in health and in inflammatory diseases.

The immune system protects from infections and cancer through complex cellular networks. For this purpose, immune cells require well-developed mechanisms of energy generation. However, the immune system itself can also cause diseases when defective regulation results in the emergence of autoreactive lymphocytes. Recent studies provide insights into how differential patterns of immune cell responses are associated with selective metabolic pathways. This review will examine the changing metabolic requirements of Th17 cells and of B cells at different stages of their development and activation. Both cells provide protection but can also mediate diseases through the production of autoantibodies and the production of proinflammatory mediators. In health, B cells produce antibodies and cytokines and present antigens to T cells to mount specific immunity. Th17 cells, on the other hand, provide protection against extra cellular pathogens at mucosal surfaces but can also drive chronic inflammation. The latter cells can also promote the differentiation of B cells to plasma cells to produce more autoantibodies. Metabolism-regulated checkpoints at different stages of their development ensure the that self-reactive B cells clones and needless production of interleukin (IL-)17 are limited. The metabolic regulation of the two cell types has some similarities, e.g. the utility of hypoxia induced factor (HIF)1α during low oxygen tension, to prevent autoimmunity and regulate inflammation. There are also clear differences, as Th17 cells only are vulnerable to the lack of certain amino acids. B cells, unlike Th17 cells, are also dependent of mechanistic target of rapamycin 2 (mTORC2) to function. Significant knowledge has recently been gained, particularly on Th17 cells, on how metabolism regulates these cells through influencing their epigenome. Metabolic dysregulation of Th17 cells and B cells can lead to chronic inflammation. Disease associated alterations in the genome can, in addition, cause dysregulation to metabolism and, thereby, result in epigenetic alterations in these cells. Recent studies highlight how pathology can result from the cooperation between the two cell types but only few have so far addressed the key metabolic alterations in such settings. Knowledge of the impact of metabolic dysfunction on chronic inflammation and pathology can reveal novel therapeutic targets to treat such diseases.

Mucosal-Associated Invariant T cells exhibit distinct functional signatures associated with protection against typhoid fever

We have previously demonstrated that Mucosal-Associated Invariant T (MAIT) cells secrete multiple cytokines after exposure to Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever in humans. However, whether cytokine secreting MAIT cells can enhance or attenuate the clinical severity of bacterial infections remain debatable. This study characterizes human MAIT cell functions in subjects participating in a wild-type S. Typhi human challenge model. Here, we found that MAIT cells exhibit distinct functional signatures associated with protection against typhoid fever. We also observed that the cytokine patterns of MAIT cell responses, rather than the average number of cytokines expressed, are more predictive of typhoid fever outcomes. These results might enable us to objectively, based on functional parameters, identify cytokine patterns that may serve as predictive biomarkers during natural infection and vaccination.

CIGB-300-Regulated Proteome Reveals Common and Tailored Response Patterns of AML Cells to CK2 Inhibition.

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

Oral shedding of herpesviruses and clinical outcomes in hematopoietic stem cell transplant patients.

To characterize the oral shedding of herpes viruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and investigate its relationship with clinical outcomes. Polymerase chain reaction and enzymatic digestion were performed to identify the oral shedding of the members of the Herpesviridae family in 31 patients. The samples were collected from the oral cavity at five timestamps. The presence of each herpesvirus in the oral cavity was observed in 3.2%, 12.9%, 19.3%, 32.2%, 54.8% and 93.5% patients for human herpesvirus (HHV)-6A, herpes simplex virus-1, HHV-6B, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and HHV-7, respectively. Oral shedding of herpes virus was not uncommon after alloHSCT. There was a statistically significant association between the EBV and CMV oral shedding at C1 and the cumulative incidence of acute graft-versus-host disease (aGVHD). The results suggested that the presence of HSV-1 at C2 was related to a relapse. The HHV-7 oral shedding at C2 suggests a possible link between relapse, progression-free survival and overall survival of the patients. Patients who developed aGVHD showed higher CMV and EBV shedding in the oral cavity at aplasia, suggesting modifications to the pattern of immune cell response and inflammatory microenvironment.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

In vitro model of distinct catabolic and inflammatory response patterns of endothelial cells to intervertebral disc cell degeneration

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-β, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1β-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1β-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.

Multidimensional Single Cell Analysis Shows Increased T/NK Cell Subsets in Both Blood and Bone Marrow of Iberdomide (CC-220) Treated Relapsed/Refractory Multiple Myeloma Patients

Background: Iberdomide (IBER), a novel cereblon (CRBN) E3 ligase modulatory compound (CELMoD), is being evaluated in a phase 1/2 clinical trial for treatment (tx) of RRMM, as a monotherapy, in combination with low-dose dexamethasone (DEX), or in combination with DEX and daratumumab, bortezomib, or carfilzomib (CC-220-MM-001; NCT02773030). IBER modulates the CRBN E3 ligase, inducing ubiquitination and proteasome-dependent degradation of Ikaros, Aiolos, and ZFP91, resulting in inhibition of myeloma-cell growth and immunomodulation. Methods: As of June 28, 2019, 95 patients (pts) had received IBER as monotherapy or in combination with DEX at doses ranging from 0.3 to 1.3 mg taken once daily (QD) on Days 1-21 of 28-day cycles (21/28). Longitudinal immune profiling by peripheral blood (PB) flow cytometry was completed at Cycle (C) 1 Day (D) 1 and C2D15 in 90 pts dosed up to 1.3 mg. T cell and NK cell proliferation was assessed by Ki67 staining and T cell activation was assessed by HLA-DR expression. T cell differentiation was assessed using markers for CD45RA, CD45RO, and CCR7. To assess immune changes in the bone marrow (BM) microenvironment, viably frozen CD138- cells from BM aspirates were obtained from pts at screening and C2D15 (10 and 6 samples available respectively; dosed from 0.3 - 0.9 mg). Samples were analyzed using both single-cell RNA sequencing (scRNA-seq) and mass cytometry by time-of-flight (CyTOF) with a novel panel designed to identify B cells, T and NK cell (sub)populations, monocytes, dendritic cells, MDSCs and Tregs. An R based workflow using the CATALYST (Crowell et al. 2019) and Seurat package (Stuart et al. Cell 2019) was used to analyze CyTOF and scRNA-seq data, respectively. Results: PB immune profiling of pts showed increases in total and proliferating NK cells following IBER tx across all doses (median increase = 35.5% and 74.2%, respectively). A dose-dependent increase in proliferating, activated and effector memory CD4+ (median increase = 92.3%, 45.5%, 51.7%, respectively, across all doses) and CD8+ (median increase = 116.0%, 13.9%, 45.8%, respectively, across all doses) T cells was also observed. To understand if these changes were concurrent with changes in the tumor microenvironment, a novel CyTOF panel was produced to analyze BM samples. Analogous to the PB findings, total BM NK cells increased (6.05% cells at screening vs. 9.91% at C2D15). CD16+ NK cells, known for their cytolytic activity, increased most prominently (35.9% of NK cells to 42.4%) vs. CD16- NK cells (64.1% of NK cells to 57.6%). Higher median CD16, CD56 and HLA-DR surface expression at C2D15 was also observed vs screening, demonstrating increased abundance and differentiation of NK cells. Similar to observations in the T cell compartment of PB, decreases in naïve CD4+ (24.4% to 9.09% of CD4+ cells) and CD8+ (6.42% to 2.14% of CD8+ cells) T cells and increases in effector memory CD4+ (35.8% to 40.1%) and CD8+ (26.7% to 39.5%) T cells were observed in BM. Transcriptomic data in BM showed increased expression of genes associated with cytolytic/cytotoxic activity (NKG7, GZMB, CCL3/4) and the interferon response pathway (IFNG, TNF, IFIT2/3) in CD8+ T cells, further supporting a shift towards activation in BM T cells with IBER tx. Conversely, we observed decreased expression of genes associated with a resting state (KLF2, ITGB7, CD52, S100A4) on CD4+ T cells in BM. Consistent with data from PB and of other immunomodulatory drugs, we also observed an increase in CD4+ regulatory T cells upon tx (0.72% to 5.06% of CD4+ T cells) in BM, however, the consequence of this observation in terms of response remains unclear. Conclusions: Pharmacodynamic profiling of immune subsets in PB and BM of RRMM pts receiving IBER showed consistent changes in NK cells and T cell subsets, suggesting increased proliferation and a more activated phenotype. Ongoing analyses will address whether such patterns of immune cell responses are more pronounced at higher doses of IBER and how they may correlate with clinical outcome. Disclosures Amatangelo: Celgene Corporation: Employment, Equity Ownership. Gooding:Celgene: Research Funding. Oppermann:Bayer Pharmaceuticals: Research Funding; GSK: Research Funding; Celgene: Research Funding. Pierceall:Celgene: Employment. Thakurta:Celgene: Employment, Equity Ownership. Parekh:Karyopharm Inc.: Research Funding; Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding.

Spike burst-pause dynamics of Purkinje cells regulate sensorimotor adaptation.

Cerebellar Purkinje cells mediate accurate eye movement coordination. However, it remains unclear how oculomotor adaptation depends on the interplay between the characteristic Purkinje cell response patterns, namely tonic, bursting, and spike pauses. Here, a spiking cerebellar model assesses the role of Purkinje cell firing patterns in vestibular ocular reflex (VOR) adaptation. The model captures the cerebellar microcircuit properties and it incorporates spike-based synaptic plasticity at multiple cerebellar sites. A detailed Purkinje cell model reproduces the three spike-firing patterns that are shown to regulate the cerebellar output. Our results suggest that pauses following Purkinje complex spikes (bursts) encode transient disinhibition of target medial vestibular nuclei, critically gating the vestibular signals conveyed by mossy fibres. This gating mechanism accounts for early and coarse VOR acquisition, prior to the late reflex consolidation. In addition, properly timed and sized Purkinje cell bursts allow the ratio between long-term depression and potentiation (LTD/LTP) to be finely shaped at mossy fibre-medial vestibular nuclei synapses, which optimises VOR consolidation. Tonic Purkinje cell firing maintains the consolidated VOR through time. Importantly, pauses are crucial to facilitate VOR phase-reversal learning, by reshaping previously learnt synaptic weight distributions. Altogether, these results predict that Purkinje spike burst-pause dynamics are instrumental to VOR learning and reversal adaptation.

The Contribution of Inflammation to Autism Spectrum Disorders: Recent Clinical Evidence.

Autism comprises a complex and heterogeneous spectrum of neurodevelopmental disorders, usually termed autism spectrum disorders (ASD). It is more prevalent in males than females, and genetic and environmental factors are believed to account in similar percentages to the development of ASD. In recent years, the contribution of inflammation and inflammatory mediators to disease aetiology and perpetuation has been the object of intense research. In this chapter, inflammatory aspects that contribute to ASD are discussed, including abnormal microglia activation and polarization phenotypes, increased systemic levels of pro-inflammatory mediators, and altered patterns of immune cell response to activation stimuli. Also, inflammation in the context of gut microbiome and the impact of inflammation on gender prevalence of ASD are considered. Finally, treatment impact on inflammatory parameters and the potential for use of anti-inflammatory drugs, alone or in combination with antipsychotics, to manage ASD are examined.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

MEK/CDK4,6 co-targeting is effective in a subset of NRAS, BRAF and 'wild type' melanomas.

Targeted therapy has become a cornerstone for the treatment of melanoma patients. Targeting NRAS function is particularly challenging. To date, only single MEK inhibitor treatment was able to show minimal clinical efficacy. The discovery that co-targeting of MEK and CDK4,6 has antitumor activity created excitement for patients and clinicians; however, it is largely unknown if only NRAS mutant patients might benefit from MEK/CDK4,6 blockade.In this study we investigate response patterns of NRAS, BRAF mutant and ‘wild type’ melanoma cells in vitro and in vivo when challenged with inhibitors of MEK, CDK4,6 and the combination of both. Data revealed, that in vitro growth response patterns of cells treated with the MEK/CDK4,6 combination correspond to in vivo efficacy of MEK/CDK4,6 co-targeting in melanoma xenograft models. Strikingly, this was consistently observed in NRAS and BRAF mutant, as well as in ‘wild type’ melanoma cells. Additionally, cells displaying elevated p-Rb levels after single MEK inhibition, showed more effective growth reduction with MEK/CDK4,6 co-targeting compared to single MEK inhibitor treatment in vivo. Findings indicate that combined MEK/CDK4,6 inhibition could offer an effectively therapeutic modality in a subset of BRAF and NRAS mutant, as well as ‘wild type’ melanoma patients.

Immunohistochemical observation of local inflammatory cell infiltration in the host-tissue reaction site of human hydatid cysts.

The aim of this study was to evaluate the pattern of local immune cell infiltration in human cystic echinococcosis (CE) by identifying the subtypes of immune cells using immunohistochemistry (IHC). Fifty surgically removed hydatid cyst samples and surrounding tissues were collected from patients referred to Al-Zahra Hospital, Isfahan, Iran. IHC was performed on the surrounding host tissue of hydatid cysts using anti-human CD3, CD19, CD8, CD4, CD68, CD56, Ki-67 and Foxp3 (forkhead box P3) antibodies. The results were then compared to hepatocellular carcinoma and chronic hepatitis. In the host-tissue reaction site of liver hydatid cysts, a distinct pattern of local immune cell response, which outwardly consisted of a pack of the fibrous elements, a layer of palisading macrophages, an eosinophil-containing layer and a layer of accumulated lymphocytes, was observed. However, in some cases there were no positive cells for CD56+ natural killer cells and Foxp3+ regulatory T cells. The CD3+ T cells were the predominant inflammatory cells in all groups, followed by CD19+ B cells. It can be concluded that different immune cells are involved in the local response to human hydatid cysts.

Comparison of T Helper Cell Patterns in Primary Open-Angle Glaucoma and Normal-Pressure Glaucoma.

BackgroundHSP60-related immunological activities are found in normal-pressure glaucoma (NPG) patients, in whom an elevated intraocular pressure (IOP) found in primary open-angle glaucoma (POAG) is not observed. HSP60 was found in POAG and NPG patients, while anti-HSP60 level was mainly found to be higher in NPG patients. The purpose of this study was to compare the percentages of Th cells and levels of related cytokines, attempting to provide evidence to explain this discrepancy.Material/MethodsBlood samples from POAG, NPG, and normal control (NC) groups were collected and peripheral blood monocytes were isolated and cultured with or without the stimulation of HSP60. Flow cytometry and enzyme-linked immunosorbent assay were used to assess the percentages of Th1, Th2, Th17, and Treg cells, as well as HSP60 antibody levels and related cytokine levels, before and after culture.ResultsSignificantly higher titers of anti-HSP60 were observed only in NPG patients. Comparable Th1 and Th2 cell frequencies, IL-4 level, and IFN-γ level were found in POAG and NPG patients, while higher Treg cell frequency was only found in POAG patients. After culturing with HSP60, increased Th2 frequencies and decreased Th1 frequencies were observed in the POAG, NPG, and NC groups, while increased Treg frequency was only identified in the POAG and NC groups.ConclusionsDifferent Th cell patterns were observed among POAG, NPG, and NC groups. Lack of induction of Treg cells and imbalance of the pro-inflammatory and anti-inflammatory response patterns of Th cells exist in some NPG patients.

Fluorescent Based Tracing of Sensory Nerve Subtypes in Brainstem

Processing vagal and trigeminal afferent inputs in the brainstem is essential for regulating reflexes including those from the respiratory tract. Afferent nerves can be categorized into several subtypes depending on the ganglionic origin of cell bodies (placodal vs. neural crest), conduction velocity and response patterns to stimuli. These various nerve subtypes convey different types of information which is integrated at the brainstem to control respiratory reflexes. Vagal afferent nerve subtypes express particular ion channels/receptors which are stimulated by various stimuli resulting in different information being relayed to the brainstem. For example, stimulation of vagal jugular C‐fiber nociceptors evokes cough, whereas stimulation of vagal nodose C‐fiber nociceptors inhibits cough. Vagal afferents are known to innervate the nucleus of the solitary tract in the dorsal medulla. However, the innervation patterns may vary depending on the subsets of vagal afferents. The variation makes it difficult to understand how distinct peripheral vagal afferent inputs are processed in the central nervous system.In the current study, we focused on developing a neuroanatomical map of various sensory afferent nerve subtypes using a transgenic mouse model system. Cre‐LoxP reporter system allows us to target the specific ion channel/receptor positive sensory nerve subtypes enabling tracking of afferent nerve subtypes throughout medulla. Transient receptor potential vanilloid 1 (Trpv1), tachykinin 1 (Tac1) and 5‐hydroxytriptamine receptor 3 (5HT3) Cre strains were bred with Floxed Ai9 ROSA tdTomato; thus, expressing red fluorescent specific to Trpv1, Tac1 and 5HT3 positive cells. Mice (6 to 8‐week‐old) were euthanized and tissue was collected. Brainstem were cryosectioned at 30 μm thickness and slide mounted sequentially. Additionally, we used a passive tissue clearing technique (CLARITY‐PACT) to visualize the sensory nerve projections in dissected whole medulla. Images were taken using Nikon Eclipse microscope and Olympus FV1200 confocal microscope. The 3D images were processed with Imaris software.As a result, we were able to identify each sensory nerve subtype in the medulla and define their projections to different regions of medulla ‐ these include the nucleus of solitary tract (NTS), paratrigeminal nucleus (Pa5) and spinal trigeminal tract (sp5). While brainstem projection of the Trpv1, Tac1 and 5ht3 Cre/Rosa strains showed similarities in gross regional expression, the patterns of these projections were distinct depending on the strains. For example, Trpv1‐tdTomato projections were found in the commissural NTS but not the medial NTS, whereas Tac1‐tdTomato projections were largely absent from the commissural NTS but were abundantly present in the medial NTS. The only overlap of Trpv1 and Tac1 projections was not in the NTS, but rather in Pa5 and sp5, suggesting that vagal jugular C‐fiber nociceptors may terminate in Pa5 as opposed to the NTS. The cleared whole medulla from each strain were consistent with the medulla sections and were used to yield 3D maps of the sensory afferent nerve projections. Based on these observations, a genetic tool using Cre‐LoxP reporter system allowed us to define the neuronal circuitry of major vagal sensory afferent nerve subtypes. This information may be useful to study functionalities of each sensory nerve subtypes to better understand the mechanism of respiratory reflexes.Support or Funding InformationNIH Common Fund SPARC OT2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape

Endotheliotropism is a hallmark of gallinaceous poultry infections with highly pathogenic avian influenza (HPAI) viruses and a feature that distinguishes HPAI from low pathogenic avian influenza (LPAI) viruses. Here, we used chicken aortic endothelial cells (chAEC) as a novel in vitro infection model to assess the susceptibility, permissiveness, and host response of chicken endothelial cells (EC) to infections with avian influenza (AI) viruses. Our data show that productive replication of AI viruses in chAEC is critically determined by hemagglutinin cleavability, and is thus an exclusive trait of HPAI viruses. However, we provide evidence for a link between limited (i.e. trypsin-dependent) replication of certain LPAI viruses, and the viruses’ ability to dampen the antiviral innate immune response in infected chAEC. Strikingly, this cell response pattern was also detected in HPAI virus-infected chAEC, suggesting that viral innate immune escape might be a prerequisite for robust AI virus replication in chicken EC.

Immune Cell Dynamics in Nordic FL Patients in the SAKK 35/10 Randomized Trial with Rituximab and Lenalidomide

Background:Follicular lymphoma (FL) is the second most common lymphoma in adults. Although responsive to various therapies it is considered incurable. The CD20 antibody rituximab is well known to have improved outcome, acting through complement-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To enhance the efficacy of rituximab different combination regimens have been used, mostly with chemotherapy but also with cytokines and immunomodulatory agents such as lenalidomide.Lenalidomide has been shown to induce durable responses with manageable toxicity in indolent lymphomas and mantle cell lymphoma. It modulates signaling pathways, enhances the capacity of T cells and natural killer (NK) cells and suppresses angiogenesis. When combined with rituximab, effects seem to be synergistic (Fowler 2014).Aim:To investigate the dynamics of immune cells in blood in patients treated with rituximab with or without lenalidomide.Patients and Methods:FL patients included in a multicenter randomized phase II trial performed by the Swiss Group for Clinical Cancer Research (SAKK) in collaboration with the Nordic Lymphoma Group (NLG) were randomized 1:1 to treatment either with rituximab alone or rituximab and lenalidomide. Inclusion criteria were histologically confirmed CD20+ FL grade 1, 2 or 3A, disease stage Ann Arbor III-IV (or II not suitable for radiotherapy) and need of therapy. In both treatment arms rituximab was administered as 4 single infusions of 375 mg/m2 weeks 1, 2, 3 and 4; patients who showed at least a minor response received 4 additional infusions at weeks 12, 13, 14 and 15. In the combination arm lenalidomide 15 mg p.o. daily was started 14 days before the first infusion and given continuously until 14 days after the last.Blood cells were sequentially sampled: at baseline, after 2 weeks’ of lenalidomide (combination arm solely), 24 hours after the first rituximab infusion and at follow-up weeks 10 and 23. Analyses of CD4+, CD8+, CD56+CD3- (NK) cells, CD19+CD20+ and CD3-CD4+ (mainly monocytes) were performed with flow cytometry and measured as percentages of mononuclear cells. T cell subpopulations CD27+CD45RA+ (naïve), CD27+CD45RO+ (memory) and CD26-CD45RA+ (effector) were measured as percentages of CD3+CD4+ and CD3+CD8+, respectively.Results:Rituximab and lenalidomide: Immune cell levels were analyzed in 13 Norwegian and Swedish patients randomized to combination treatment. Compared to baseline, the fraction of CD3-CD4+ cells increased in all but two (one of whom had an additional population of CLL cells), accompanied by decrease in CD4+ cells. As previously described NK cell levels rose (ASH 2015 abstract 1535), here in 9/12 patients. In 8/12 the CD4/CD8 ratio decreased.Among CD4+ effector cells were few at baseline. Among CD8+ they were more abundant but decreased with lenalidomide. In both CD4+ and CD8+, naïve cells were reduced in favor of memory cells.7 patients out of these 13 had blood samples retrieved 24h after first rituximab infusion. The B cell depletion initiated already with lenalidomide, was deepened. The NK cell fraction increased, as did fractions of naïve T cells, for CD4+ in 7/7 patients and for CD8+ in 6/7.Figure 1: Average changes in immune cell composition among 7 patients treated with lenalidomide and subsequent rituximab.Single rituximab: Among 7 rituximab treated patients sampled both at baseline and 24h post rituximab, NK cells decreased markedly in all but one. B cells were reduced as predicted. In comparison with 7 patients treated with rituximab and lenalidomide, the combination arm showed a relatively larger fraction of memory T cells.Conclusion:Changes in immune cell subsets were found in peripheral blood of FL patients after rituximab with or without lenalidomide. After lenalidomide alone, T cells developed from naïve to memory stages, and an increase in monocyte and NK cell fractions was found, the latter decreasing again after first rituximab as previously seen. Between treatment arms differences in memory T-cells was seen also after the first rituximab dose. Clinical outcome in relation to patterns of immune cell response is under analysis. [Display omitted] DisclosuresWahlin:Roche: Consultancy. Kimby:Gilead: Honoraria; Baxalta: Consultancy; Abbvie: Consultancy, Honoraria; Jansen: Consultancy, Honoraria; Celgene: Honoraria.