Immunology Studies Research Articles

Plant immunity and sustainable control of pests in China: Advances, opportunities and challenges

Plant immunology studies interactions between plants and pathogenic organisms. Like animals, plants possess a powerful innate immune system, which recognizes pathogenic organisms including viruses, bacteria, fungi, oomycetes, nematodes, insects and parasitic weeds to mount a series of defenses to fend off invading pathogens. Pathogens can evade or overcome host immune systems to cause diseases and damage on host plants. Over the last two decades, China has made great strides in plant immunity research and contributed significantly to multiple areas of studies. These advances shed new light on the development of effective technologies for sustainable control of pests in agriculture.

The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: a 6-cysteine protein family member.

IntroductionThe zoonotic malaria parasite Plasmodium knowlesi has currently become the most dominant form of infection in humans in Malaysia and is an emerging infectious disease in most Southeast Asian countries. The P41 is a merozoite surface protein belonging to the 6-cysteine family and is a well-characterized vaccine candidate in P. vivax and P. falciparum; however, no study has been done in the orthologous gene of P. knowlesi. This study investigates the level of polymorphism, haplotypes and natural selection of pk41 genes in clinical isolates from Malaysia.MethodThirty-five full-length pk41 sequences from clinical isolates of Malaysia along with four laboratory lines (along with H-strain) were downloaded from public databases. For comparative analysis between species, orthologous P41 genes from P. falciparum, P. vivax, P. coatneyi and P. cynomolgi were also downloaded. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. Phylogenetic relationships between Pk41 genes were determined using MEGA 5.0 software.ResultsAnalysis of 39 full-length pk41 sequences along with the H-strain identified 36 SNPs (20 non-synonymous and 16 synonymous substitutions) resulting in 31 haplotypes. Nucleotide diversity across the full-length gene was low and was similar to its ortholog in P. vivax; pv41. Domain-wise amino acid analysis of the two s48/45 domains indicated low level of polymorphisms for both the domains, and the glutamic acid rich region had extensive size variations. In the central domain, upstream to the glutamate rich region, a unique two to six (K-E)n repeat region was identified within the clinical isolates. Overall, the pk41 genes were indicative of negative/purifying selection due to functional constraints. Domain-wise analysis of the s48/45 domains also indicated purifying selection. However, analysis of Tajima’s D across the genes identified non-synonymous SNPs in the s48/45 domain II with high positive values indicating possible epitope binding regions. All the 6-cysteine residues within the s48/45 domains were conserved within the clinical isolates indicating functional conservation of these regions. Phylogenetic analysis of full-length pk41 genes indicated geographical clustering and identified three subpopulations of P. knowlesi; one originating in the laboratory lines and two originating from Sarawak, Malaysian Borneo.ConclusionThis is the first study to report on the polymorphism and natural selection of pk41 genes from clinical isolates of Malaysia. The results reveal that there is low level of polymorphism in both s48/45 domains, indicating that this antigen could be a potential vaccine target. However, genetic and molecular immunology studies involving higher number of samples from various parts of Malaysia would be necessary to validate this antigen’s candidacy as a vaccine target for P. knowlesi.

Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning.

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

SELF OR NON-SELF? CONSTRUCTING THE BODY IN IMMUNOLOGY

The article focusses on how the body is constructed for immunology. Specifically, the complex and tangled bio-political genealogy of immunity is analysed in detail as the understanding of it hovers between social and biological discourses. In addition, the controversy between different models of the immune system is examined. The social studies of immunology conducted by Donna Haraway and Emily Martin are also highlighted. In these studies, the body-immune system is understood as a diversified heterogeneous construction consisting of components belonging to different ontological orders. The construction of the immune system does not end in the labora-tory or in the clinic. It continues in other places by other means. Nevertheless, the relationship between the body and the immune system is situational: the immune system could be completely identified with the body or be a part of it. Emily Martin’s ethnography of immune systems and Donna Haraway’s feminist anthropology provide the means for understanding how the immune system, as both academics and non-academics explain it, can be juxtaposed to and also coincide with the body and even the self,but nevertheless conform to the scale, concepts, laws and metaphors of the social world of everyday life. The immune system and immunity are assessed in terms of the physiological body and the self. On the other hand, the body as a biological “self” is abstracted from the physiological and social body, and in a sense it “lives a life of its own.” Therefore, our body is the outcome of a complex coordinating effort among different bodies: the physiological body, the one identified with the self, and the body on a different scale - the biological “self” which turns out to be “non-self” and not belong to the subject

PH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State

The recent spread of Zika virus stimulated extensive research on its structure, pathogenesis, and immunology, but mechanistic study of entry has lagged behind, in part due to the lack of a defined reconstituted system. Here, we report Zika membrane fusion measured using a platform that bypasses these barriers, enabling observation of single-virus fusion kinetics without receptor reconstitution. Surprisingly, target membrane binding and low pH are sufficient to trigger viral hemifusion to liposomes containing only neutral lipids. Second, although the extent of hemifusion strongly depends on pH, hemifusion rates are relatively insensitive to pH. Kinetic analysis shows that an off-pathway state is required to capture this pH-dependence and suggests this may be related to viral inactivation. Our surrogate-receptor approach thus yields new understanding of flaviviral entry mechanisms and should be applicable to many emerging viruses.

Characterization of an NLRP1 Inflammasome from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory Caspases in Ancient Vertebrates.

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, Danio rerio NLRP1 (DrNLRP1) from a zebrafish (D. rerio) model. This DrNLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases (D. rerio Caspase [DrCaspase]-A and DrCaspase-B) and maturation of D. rerio IL-1β in a D. rerio ASC (DrASC)-dependent manner. In this process, DrNLRP1 promotes the aggregation of DrASC into a filament with DrASCCARD core and DrASCPYD cluster. The assembly of DrNLRP1 inflammasome depends on the CARD-CARD homotypic interaction between DrNLRP1 and DrASCCARD core, and PYD-PYD interaction between DrCaspase-A/B and DrASCPYD cluster. The FIIND domain in DrNLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two DrCaspases are coordinated in DrNLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation of DrCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for DrCaspase-A followed by a subsequent selection for DrCaspase-B. By using morpholino oligonucleotide-based knockdown assays, the DrNLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.

A model for tumor-immune interaction.

Studies of tumor immunology will gain from coculture of tumor organoids and peripheral blood cells.

Evaluation of the reproductive potential of Encephalitozoon intestinalis in four different cell line

Microsporidia are parasites that can cause infections in many vertebrate and invertebrate organisms and produce small spores resistant to environmental conditions. As they are obligate intracellular parasites, axenic cultures cannot be performed. The aim of this study was to investigate the reproductive potential of the parasite in human colon epidermal adenocarcinoma (Caco-2), human monocytic (U937), African green monkey renal epithelial (VERO) and human kidney epithelial (HEK-293) cell lines of tissue and organs where the parasite is located by following the culture of the parasites and the amount of spores for six weeks. RPMI-1640 medium was used for the cultivation of U937 cells, while DMEM was used for other cell lines and the immature U937 cells were stimulated with Phorbol-12-Myristate-13-Acetate before infection. All of the host cell groups were infected with freshly collected Encephalitozoon intestinalis spores in ratio 1:30 and free spores in the culture media were removed after overnight incubation at 37°C under 5% CO2 condition for parasite invasion. The first release of the spores from the infected cells was observed and recorded by following for six weeks. Furthermore, the spore density released from each cell groups was evaluated by measuring the parasite load by Thoma cell counting chamber and quantified by real-time PCR. As a result of the study, it was observed that four cell lines could be infected by E.intestinalis and the spore production can be maintained for six weeks. It was observed that the monolayer macrophages and CaCo-2 cells, started to be detached from the culture flasks in few days following the parasite invasion, thus decreasing the number of host cells. After 1-2 weeks, HEK-293 cells were also detached from the surface, thus negatively affected the pure spore production by contaminating the media with dead host cell suspension. Spores started to appear in VERO cell media at the end of the second week after initial infection, while it took longer time for other cells to start releasing spores. Over the course of six weeks, the VERO cell line had the highest spore-producing potential among the other cell lines. In conclusion, this study compared the potential for reproduction of E.intestinalis in three human cell lines and monkey originated VERO cell line. This study demonstrated that cells derived from the tissues or organs where Microsporidia species causes disseminated infections could be infected by the parasitic spores in vitro. Additionally, the parasite can survive and propagate longer than six weeks. The authors believe that the results of this study will contribute to the further studies related to the parasite in the area of genetics, pharmacology, biochemistry, immunology and eradication studies.

Micro-patterned endogenous stroma equivalent induces polarized crypt-villus architecture of human small intestinal epithelium

The small intestine is the major site for digestion, drug and nutrient absorption, as well as a primary site for many diseases. Current in vitro gut models fail in reproducing the complex intestinal extracellular matrix (ECM) network of the lamina propria and the peculiar architecture of the crypt-villus axis. Here we proposed a novel in vitro human intestine model that mimics the intestinal stromal topography and composition and strictly reproduces the tissue polarity with the crypt-villus architecture. First we developed a 3D human intestinal stromal equivalent (3D-ISE) composed of human intestinal subepithelial myofibroblasts (ISEMFs) embedded in their own extracellular matrix. Then, we seeded human colon carcinoma-derived cells (Caco-2) onto flat or patterned cell-synthetized stromal equivalent structure and cultured them until the formation of a well-oriented epithelium. We demonstrated that the patterned stroma increases the absorbing surface area, the epithelial proliferation rate, and the density of microvilli. In addition it induces changes in the biological functions of the epithelial cells such as enzymes and mucus production, polarization and tightness showing a physiological cell-lineage compartmentalization along the crypt/villi axes with the undifferentiated phenotypes at the base. At last, we reproduced an inflamed intestinal tissue model in which we identified the contribution of the stromal microenvironment by molecular (cytokines release and MMPs production) and immunofluorescence analyses and the effects of the epithelial-stromal cross-talk in the intestinal innate immunity by multiphoton investigation that revealed differences in the collagen network architecture. Statement of SignificanceThe intestinal stroma morphology and composition has a fundamental role in crypt-villus development and appropriate epithelial cell-lineage compartmentalization. On this base, here we develop an engineered organotypic model of human intestine equivalent in which a functional epithelial/ECM crosstalk is recapitulated. Due to its accessible luminal surface it provides a new platform for preclinical studies of mucosal immunology and bowel inflammation as well as the assessment of pharmaco-toxicity studies.

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

A single centre phase II trial to assess the immunological activity of TroVax® plus pemetrexed/cisplatin in patients with malignant pleural mesothelioma – the SKOPOS trial

ABSTRACTVaccines in combination with chemotherapy have been shown to be safe in different tumor types. We investigated the immunological activity of the TroVax® vaccine in combination with pemetrexed-cisplatin chemotherapy in malignant pleural mesothelioma (MPM).In this first line, open-label, single-arm, phase 2 study, patients with locally advanced or metastatic MPM were enrolled. Eligible patients received up to 9 intramuscular injections of TroVax®, starting two weeks before chemotherapy and continuing at regular intervals during and after chemotherapy to 24 weeks. The primary endpoint was the induction of cellular or humoral anti-5T4 immune response (defined as a doubling of either response at any of six follow-up time points), with a target response rate of 64%.Of 27 patients, enrolled between Feb 2013-Dec 2014, 23 (85%) received at least three doses of TroVax® and one cycle of chemotherapy and were included in the per-protocol analysis (PPA). 22/23 patients (95.6%) developed humoral or cellular immune response to 5T4. Thus, the study reached its primary endpoint. Disease control was observed in 87% of patients (partial response: 17.4%, stable disease: 69.6%). The median progression-free survival was 6.8 months and median overall survival 10.9 months. Treatment-related adverse events were comparable to those observed in patients with chemotherapy alone. Translational immunology studies revealed a circulating baseline immune signature that was significantly associated with long-term (>20 months in n = 8/23, 34.8%) survival.In this phase 2 trial, TroVax® with pemetrexed-cisplatin chemotherapy showed robust immune activity, acceptable safety and tolerability to warrant further investigation in a phase 3 setting.

Abstract B29: Multiplex three-dimensional optical mapping of tumor immune microenvironment

Abstract Recent advances in optical tissue clearing and microscopic imaging have facilitated three-dimensional (3D) visualization of large tissues and whole organs at a high resolution. However, to expand its applications to oncology, current challenges such as slow and complicated tissue clearing and uneven immunostaining must be addressed. Here we describe transparent tissue tomography (T3), a tool for rapid, three-dimensional, multiplex immunofluorescence tumor imaging. Cutting tumors into 400 μm macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous programmed death-ligand 1 (PD-L1) distribution in transgenic mouse mammary tumors, with high expression limited to Her2+ tumor cells at the periphery and to CD31+ vascular endothelium in the core. Also, strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression at the tumor margin was determined by T3 analysis of the whole tumors. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 to immunolocalization of cytotoxic lymphocytes in whole core needle biopsies obtained from mouse mammary tumors and human head & neck tumors. We spatially and quantitatively map granzymeB-producing CD3+CD8+ cytotoxic T cells and CD49b+CD31+ natural killer cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by chromogenic immunohistochemistry (IHC). We anticipate that T3 can be applied broadly to facilitate preclinical studies of tumor immunology and immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy. Citation Format: Steve Seung-Young Lee, Vytautas Bindokas, Stephen Kron. Multiplex three-dimensional optical mapping of tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B29.

Modeling Tumor Immunology and Immunotherapy in Mice

Figure 1. Main Applications of Mouse Models for Tumor Immunology and Immunotherapy. Immunodeficient mice xenografted with human cancer cell lines have been at the foundation of in vivo cancer research for several decades, providing ground for the regulatory approval of multiple chemotherapeutics and targeted anticancer agents, but are intrinsically unsuitable for studying tumor immunology and immunotherapy. Similarly, patient-derived xenografts (PDXs) established in immunodeficient mice are not subjected to immunosurveillance by the host, although (depending on the protocol employed for PDX generation) some components of the patient's immune system may also be transferred to the mouse and be active, at least for some time. Considerable efforts are being devoted to the generation of humanized mice to circumvent these limitations. The establishment of PDXs in immunodeficient mice that are also engrafted with matched patient-derived peripheral blood mononuclear cells (PBMCs) is also being investigated as a means to screen for the efficacy of (immuno)therapeutic agents in support of clinical decision making. Mouse cancer cell lines grafted subcutaneously or orthotopically in immunocompetent syngeneic hosts have been instrumental for the development of a variety of immunotherapeutics, as well as for the discovery that conventional anticancer regimens, including some forms of chemotherapy and radiation therapy, can trigger tumor-targeting immune responses. Carcinogen-driven tumors established in immunocompetent versus immunodeficient animals were critical in the early days of modern tumor immunology, as they enabled the discovery of natural anticancer immunosurveillance. Moreover, they allow for investigating the immunological versus non-immunological efficacy of anticancer (immuno)therapeutics in the context of natural immunoediting, clinically relevant immunobiological heterogeneity, and high mutation load. Transgene-driven tumors have generated in-depth insights into the crosstalk between oncogenic drivers and the tumor microenvironment, in both its immunological and non-immunological components. Each of these models is associated with specific advantages and disadvantages (see Key Facts). Figure 2. Key Features of Mouse Models for Tumor Immunology and Immunotherapy. Key parameters that should be taken into careful consideration when choosing the most appropriate mouse model for the study of tumor immunology and immunotherapy include not only the immunological competence of the host (which is influenced by strain, sex, and age) and its immunological compatibility with malignant cells (which is dictated by strain), but also the mutational load of the latter, their immunological history (previous immunoediting), proliferative potential, propensity for neovascularization and metastatic dissemination, as well as their ability to generate an immunostimulatory versus immunosuppressive microenvironment. Inoculation site is also an important parameter to keep under consideration (not shown). The precise objective of each study dictates which specific combination of such features should be preferred.

Neonate-omics: Charting the Unknown Immune Response in Early Life

The study of neonatal immunology has been hampered by lack of access to infant samples. Novel sample sparing methods and systems-wide approaches have uniquely expanded this field, demonstrating that newborn immunity varies widely but converges over the first 3months of life. During this important time window, environmental and genetic factors impact the infant immune system and can influence lifelong immunity.

Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.

Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2′-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.

Research Progress in Biological Studies of Schizophrenia in China in 2017.

SummarySchizophrenia is a severe mental disorder and its etiology and pathological mechanism are unknown. This article mainly introduces the progress of biological studies of schizophrenia in China in 2017, including neuroimaging, genetics, and immunology studies. It also introduces the research progress of high-risk psychotic syndrome and physiotherapy.

Evaluation of the effect of chickenpox vaccination on shingles epidemiology using agent-based modeling.

BackgroundBiological interactions between varicella (chickenpox) and herpes zoster (shingles), two diseases caused by the varicella zoster virus (VZV), continue to be debated including the potential effect on shingles cases following the introduction of universal childhood chickenpox vaccination programs. We investigated how chickenpox vaccination in Alberta impacts the incidence and age-distribution of shingles over 75 years post-vaccination, taking into consideration a variety of plausible theories of waning and boosting of immunity.MethodsWe developed an agent-based model representing VZV disease, transmission, vaccination states and coverage, waning and boosting of immunity in a stylized geographic area, utilizing a distance-based network. We derived parameters from literature, including modeling, epidemiological, and immunology studies. We calibrated our model to the age-specific incidence of shingles and chickenpox prior to vaccination to derive optimal combinations of duration of boosting (DoB) and waning of immunity. We conducted paired simulations with and without implementing chickenpox vaccination. We computed the count and cumulative incidence rate of shingles cases at 10, 25, 50, and 75 years intervals, following introduction of vaccination, and compared the difference between runs with vaccination and without vaccination using the Mann–Whitney U-test to determine statistical significance. We carried out sensitivity analyses by increasing and lowering vaccination coverage and removing biological effect of boosting.ResultsChickenpox vaccination led to a decrease in chickenpox cases. The cumulative incidence of chickenpox had dropped from 1,254 cases per 100,000 person-years pre chickenpox vaccination to 193 cases per 100,000 person-years 10 years after the vaccine implementation. We observed an increase in the all-ages shingles cumulative incidence at 10 and 25 years post chickenpox vaccination and mixed cumulative incidence change at 50 and 75 years post-vaccination. The magnitude of change was sensitive to DoB and ranged from an increase of 22–100 per 100,000 person-years at 10 years post-vaccination for two and seven years of boosting respectively (p < 0.001). At 75 years post-vaccination, cumulative incidence ranged from a decline of 70 to an increase of 71 per 100,000 person-years for two and seven years of boosting respectively (p < 0.001). Sensitivity analyses had a minimal impact on our inferences except for removing the effect of boosting.DiscussionOur model demonstrates that over the longer time period, there will be a reduction in shingles incidence driven by the depletion of the source of shingles reactivation; however in the short to medium term some age cohorts may experience an increase in shingles incidence. Our model offers a platform to further explore the relationship between chickenpox and shingles, including analyzing the impact of different chickenpox vaccination schedules and cost-effectiveness studies.

Activating natural killer (NK) cytotoxicity of canine CD5-CD21- cells requires low surface CD5 density NK cells.

Natural killer (NK) cells play a crucial role in regulating immune functions. Few studies have characterized canine NK cells. We previously demonstrated that canine peripheral blood lymphocytes (PBLs) with a low surface CD5 density (CD5lo) are considered a critical NK population. Natural killer cells in most mammals do not express T-cell markers, but canine CD5lo cells express surface molecules, such as CD3 T-cell receptors. These features make canines unique models for the study of comparative immunology in NK cells. In this study, we discovered that CD5lo and CD21 double-negative (CD5lo-ne/CD2-) cells were originally low in NK cytotoxicity and their NK cytotoxicity was highly activated when co-cultured with CD5lo NK cells. The cytotoxicity was not activated when co-cultured with other cell types, such as high surface CD5 density (CD5hi) cells. The CD5lo-negative (CD5lo-ne) population comprises CD5- and CD5hi cells. CD5-cells were low in NK cytotoxicity initially or after culturing with interleukin-2 (IL-2) without CD5lo cells; however, the addition of CD5lo cells in a similar medium markedly enhanced the NK activity. By contrast, CD5hi cells were always NK inactive, irrespective of them being cultured with CD5lo cells or not. We further verified that only the CD5-CD21- cells, which were separated from CD5-CD21+ cells in the entire CD5- population, showed activated NK activity through CD5lo cell induction. This study is the first to reveal that canine NK cells enhanced NK-inert cells to become NK-cytotoxic cells. Additionally, it is concluded that in beagles, except for CD5lo cells, CD5-CD21- cells show NK activity.