Bioreactor-Based Antigen Production Process Using the Baculovirus Expression Vector System.

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.

Combining the advantages of prokaryotic expression and T7 phage display systems to obtain antigens for antibody preparation.

The gene encoding the phage major capsid protein 10A was cloned into the prokaryotic expression vector pET24a, and a 6XHis-tag was fused to the 3'-end of the 10A gene to verify complete expression. The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and 10A expression was induced by IPTG. SDS-PAGE and Western blot were used to confirm the target protein expression. The T7Select10-3b vector was added to the cultured bacteria expressing 10A at a multiplicity of infection (MOI) ranging from 0.01 to 0.1, and complete lysis of the bacteria was monitored by absorbance changes in the medium. The recombinant phage (reP) was harvested by PEG/NaCl sedimentation and resuspended in PBS. ELISA was performed to verify the presence of the 6XHis-tag on the surface of reP. The 10A-fusion expression vectors (pET10A-flag, pET10A-egfp, and pET10A-pct) were constructed, and fusion proteins were expressed and detected by the same method. The corresponding rePs (reP-Flag, reP-EGFP, and reP-PCT) were prepared by T7Select10-3b infection. After the expression of the peptides/proteins on the reP surfaces was confirmed, reP-Flag and reP-PCT were used to immunize mice to prepare anti-Flag and anti-PCT antibodies. The results showed that rePs prepared using the 10A-fusion vector and T7Select10-3b can be used as antigens to immunize mice and prepare antibodies. This method may be able to meet the rapid antigen preparation requirements for antibody production. Notably, the recombinant phage (reP) described in this study was obtained by the sedimentation method from T7Select10-3b-infected E. coli BL21 (DE3) cells carrying the major capsid protein 10A expression vector or 10A-fusion protein vector.

Selective Elimination of NG2-Expressing Hair Follicle Stem Cells Exacerbates the Sensitization Phase of Contact Dermatitis in a Transgenic Rat Model.

The hair cycle consists of three different phases: anagen (growth), catagen (regression), and telogen (resting). During the anagen phase, hair follicle stem cells (HFSCs) in the bulge and the secondary hair germ proliferate and generate the outer and inner root sheath cells and the hair shafts. We previously identified NG2-immunoreactive (NG2+) cells as HFSCs in both regions of the hair follicles. Recently, the interaction between the hair cycle and the cutaneous immune system has been re-examined under physiological and pathological conditions. However, the roles of NG2+ HFSCs in the skin's immune system remain completely elucidated. In the present study, we investigated whether the elimination of NG2+ HFSCs affects the induction of allergic contact dermatitis, using a herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene system. When the GCV solution was applied to the skin of NG2-HSVtk transgenic (Tg) rats during the depilation-induced anagen phase, NG2+ HFSCs in the Tg rat skin induced apoptotic cell death. Under exposure of a hapten, the selective ablation of NG2+ HFSCs during the anagen phase aggravated the sensitization phase of allergic contact dermatitis. These findings suggest that NG2+ HFSCs and their progeny have immunosuppressive abilities during the anagen phase.

The induction of Neuron-Glial2 (NG2) expressing cells in methamphetamine toxicity-induced neuroinflammation in rat brain are averted by melatonin.

Neuron-Glial2 (NG2) expressing cells are described as the oligodendrocyte precursor cells in the brain. This study aimed to investigate the possible involvement of NG2 cells under the methamphetamine (METH)-induced neurotoxicity and neuroprotective capacity of melatonin. The results showed that the levels of NG2 in rat brain gradually increase from postnatal day 0 to postnatal day 8 and then the lower levels of NG2 are shown in adults. In adult rats, the levels of NG2 and COX-2 in the brain were significantly increased in lipopolysaccharide treatment. Pretreatment of 10 mg/kg melatonin prior to treating with METH was able to reduce an increase in the levels of NG2 and activation in astrocyte and microglia. These findings would extend the contribution of NG2 expressing cells in the adult brain during pathological conditions such as neuroinflammation.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays.

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Rapid Antigen and Antibody-Like Molecule Discovery by Staphylococcal Surface Display.

Ever since the discovery of antibodies, they have been generated by complicated multi-step procedures. Typically, these involve sequencing, cloning, and screening after expression of the antibodies in a suitable organism and format. Here, a staphylococcal nanobody display is described that omits many the abovementioned intermediate steps and allows for simultaneous screening of multiple targets without prior knowledge nor expression of the binders. This paper reports a detailed, general step-by-step protocol to achieve nanobodies of high affinity. Apart from its focus on radioactive and fluorescent targets, it gives options for various other target formats and additional applications for the staphylococcal library; including flow cytometry and immunoprecipitation. This provides a system for antibody engineers that can be easily adopted to their specific needs.

Nestin+NG2+ Cells Form a Reserve Stem Cell Population in the Mouse Prostate.

In the prostate, stem and progenitor cell regenerative capacities have been ascribed to both basal and luminal epithelial cells. Here, we show that a rare subset of mesenchymal cells in the prostate are epithelial-primed Nestin-expressing cells (EPNECs) that can generate self-renewing prostate organoids with bipotential capacity. Upon transplantation, these EPNECs can form prostate gland tissue grafts at the clonal level. Lineage-tracing analyses show that cells marked by Nestin or NG2 transgenic mice contribute to prostate epithelium during organogenesis. In the adult, modest contributions in repeated rounds of regression and regeneration are observed, whereas prostate epithelial cells derived from Nestin/NG2-marked cells are dramatically increased after severe irradiation-induced organ damage. These results indicate that Nestin/NG2 expression marks a novel radioresistant prostate stem cell that is active during development and displays reserve stem cell activity for tissue maintenance.

Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice.

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-ß (TGF-ß) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.

Early embryonic NG2 glia are exclusively gliogenic and do not generate neurons in the brain.

In the central nervous system, the type I transmembrane glycoprotein NG2 (nerve-glia antigen 2) is only expressed by pericytes and oligodendrocyte precursor cells (OPCs). Therefore, OPCs are also termed NG2 glia. Their fate during development has been investigated systematically in several genetically modified mouse models. Consensus exists that postnatal NG2 glia are restricted to the oligodendrocyte (OL) lineage, while, at least in the forebrain, embryonic NG2 glia could also generate astrocytes. In addition, experimental evidence for a neurogenic potential of NG2 glia in the early embryonic brain (before E16.5) has been provided. However, this observation is still controversial. Here, we took advantage of reliable transgene expression in NG2-EYFP and NG2-CreERT2 knock-in mice to study the fate of early embryonic NG2 glia. While pericytes were the main cells with robust NG2 gene activity at E12.5, only a few OPCs expressed NG2 at this early stage of embryogenesis. Subsequently, this proportion of OPCs increased from 3% (E12.5) to 11% and 25% at E14.5 and E17.5, respectively. When Cre DNA recombinase activity was induced at E12.5 and E14.5 and pups were analyzed at postnatal day 0 (P0) and P10, the vast majority of recombined cells, besides pericytes, belonged to the OL lineage cells, with few astrocytes in the ventral forebrain. In other brain regions such as brain stem, cerebellum, and olfactory bulb only OL lineage cells were detected. Therefore, we conclude that NG2 glia from early embryonic brain are restricted to a gliogenic fate and do not differentiate into neurons after birth.

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells.

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.

Injury type-dependent differentiation of NG2 glia into heterogeneous astrocytes.

The glial scar is comprised of a heterogeneous population of reactive astrocytes. NG2 glial cells (also known as oligodendrocyte progenitor cells or polydendrocytes) may contribute to this heterogeneity by differentiating into astrocytes in the injured CNS, but there have been conflicting reports about whether astrocytes comprise a significant portion of the NG2 cell lineage. By using genetic fate mapping after spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) in mice, the goal of this study was to confirm and extend upon previous findings, which have shown that NG2 cell plasticity varies across CNS injuries. We generated mice that express tdTomato in NG2 lineage cells and express GFP under the Aldh1l1 or Glt1 promoter so that NG2 glia-derived astrocytes can be detected by their expression of GFAP and/or GFP. We found that astrocytes comprise approximately 25% of the total NG2 cell lineage in the glial scar by 4 weeks after mid-thoracic contusive SCI, but only 9% by the peak of functional deficit after EAE. Interestingly, a subpopulation of astrocytes expressed only GFP without co-expression of GFAP, uncovering their heterogeneity and the possibility of an underestimation of NG2 glia-derived astrocytes in previous studies. Additionally, we used high performance liquid chromatography to measure the level of tamoxifen and its metabolites in the spinal cord and show that genetic labeling of NG2 glia-derived astrocytes is not an artifact of residual tamoxifen. Overall, our data demonstrate that a heterogeneous population of astrocytes are derived from NG2 glia in an injury type-dependent manner.

Baculovirus Surface Display of Immunogenic Proteins for Vaccine Development.

Vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. To date, several viral vectors have been utilized for the generation of vaccines. Among them, baculovirus-categorized as a nonhuman viral vector-has been used in wider applications. Its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. In addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. Furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. Herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens.

Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

[Advances in chloroplast expression of recombinant proteins in higher plants].

In recent years, gene engineering is developing rapidly and many recombinant proteins have been expressed. The use of plant bioreactor to express specific pharmaceutical proteins provides a new way for the prevention and treatment of some important diseases in human beings. Nowadays, chloroplast genetic transformation and expression system has become a research hotspot in plant bioreactor. Higher plant chloroplasts have unique advantages in the expression of recombinant proteins due to their special structures and inherited characteristics: such as high expression, site-specific integration, and the maternal inheritance characteristics of exogenous genes. The maternal inheritance of chloroplast is helpful for biological safety of transgene escaping by pollens. Many important pharmaceutical proteins have been successfully expressed in plant chloroplasts. As a chloroplast transformation model of higher plants, tobacco has made significant progress in the expression of pharmaceutical proteins, such as vaccine antigens, antibodies, and other important recombinant proteins. Chloroplast genetic transformation in higher plants also provides new techniques and methods for the study of chloroplast gene expression and regulation mechanisms. In order to provide a new idea for the development of chloroplast expression platform and the expression of important pharmaceutical proteins, this review outlined the progress of chloroplast genetic transformation system in higher plants, including the chloroplast transformation principle, vector construction, expression of recombinant proteins and important pharmaceutical proteins, and the effects of recombinant proteins expression on plant metabolism and traits.

Male germ cells support long-term propagation of Zika virus.

Evidence of male-to-female sexual transmission of Zika virus (ZIKV) and viral RNA in semen and sperm months after infection supports a potential role for testicular cells in ZIKV propagation. Here, we demonstrate that germ cells (GCs) are most susceptible to ZIKV. We found that only GCs infected by ZIKV, but not those infected by dengue virus and yellow fever virus, produce high levels of infectious virus. This observation coincides with decreased expression of interferon-stimulated gene Ifi44l in ZIKV-infected GCs, and overexpression of Ifi44l results in reduced ZIKV production. Using primary human testicular tissue, we demonstrate that human GCs are also permissive for ZIKV infection and production. Finally, we identified berberine chloride as a potent inhibitor of ZIKV infection in both murine and human testes. Together, these studies identify a potential cellular source for propagation of ZIKV in testes and a candidate drug for preventing sexual transmission of ZIKV.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice.

Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2KO). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2KO OPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2KO animals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2KO OPCs were identified by a Cre reporter, we establish that Cav1.2KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca2+ channel for OPC maturation during the remyelination of the adult brain.SIGNIFICANCE STATEMENT Ion channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca2+ entry is involved in oligodendrocyte differentiation and remyelination, we used a conditional knockout mouse for VGCCs in OPCs. Our results indicate that VGCCs can modulate oligodendrocyte maturation in the demyelinated brain and suggest that voltage-gated Ca2+ influx in OPCs is critical for remyelination. These findings could lead to novel approaches for obtaining a better understanding of the factors that control OPC maturation in order to stimulate this pool of progenitors to replace myelin in demyelinating diseases.

Designer outer membrane vesicles as immunomodulatory systems - Reprogramming bacteria for vaccine delivery.

Vaccines often require adjuvants to be effective. Traditional adjuvants, like alum, activate the immune response but in an uncontrolled way. Newer adjuvants help to direct the immune response in a more coordinated fashion. Here, we review the opportunity to use the outer membrane vesicles (OMVs) of bacteria as a way to modulate the immune response toward making more effective vaccines. This review outlines the different types of OMVs that have been investigated for vaccine delivery and how they are produced. Because OMVs are derived from bacteria, they have compositions that may not be compatible with parenteral delivery in humans; therefore, we also review the strategies brought to bear to detoxify OMVs while maintaining an adjuvant profile. OMV-based vaccines can be derived from the pathogens themselves, or can be used as surrogate constructs to mimic a pathogen through the heterologous expression of specific antigens in a desired host source strain, and approaches to doing so are reviewed. Additionally, the emerging area of engineered pathogen-specific carbohydrate sequences, or glycosylated OMVs is reviewed and contrasted with protein antigen delivery. Existing OMV-based vaccines as well as their routes of administration round out the text. Overall, this is an exciting time in the OMV field as it matures and leads to more effective and targeted ways to induce desired pathogen-specific immune responses.

A novel excision selection method for isolation of antibodies binding antigens expressed specifically by rare cells in tissue sections.

There is a growing appreciation of single cell technologies to provide increased biological insight and allow development of improved therapeutics. The central dogma explains why single cell technologies is further advanced in studies targeting nucleic acids compared to proteins, as nucleic acid amplification makes experimental detection possible. Here we describe a novel method for single round phage display selection of antibody fragments from genetic libraries targeting antigens expressed by rare cells in tissue sections. We present and discuss the results of two selections of antibodies recognizing antigens expressed by perivascular cells surrounding capillaries located in a human brain section; with the aim of identifying biomarkers expressed by pericytes. The area targeted for selection was identified by a known biomarker and morphological appearance, however in situ hybridizations to nucleic acids can also be used for the identification of target cells. The antibody selections were performed directly on the tissue sections followed by excision of the target cells using a glass capillary attached to micromanipulation equipment. Antibodies bound to the target cells were characterized using ELISA, immunocytochemistry and immunohistochemistry. The described method will provide a valuable tool for the discovery of novel biomarkers on rare cells in all types of tissues.

Mining a differential sialotranscriptome of Rhipicephalus microplus guides antigen discovery to formulate a vaccine that reduces tick infestations.

BACKGROUND: Ticks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines. RESULTS: Different levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them. CONCLUSION: Mining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.

Amylin alters human brain pericyte viability and NG2 expression.

Amylin, a pancreatic ß-cell-derived peptide hormone, forms inclusions in brain microvessels of patients with dementia who have been diagnosed with type 2 diabetes and Alzheimer's disease. The cellular localization of these inclusions and the consequences thereof are not yet known. Using immunohistochemical staining of hippocampus and parahippocampal cortex from patients with Alzheimer's disease and non-demented controls, we show that amylin cell inclusions are found in pericytes. The number of amylin cell inclusions did not differ between patients with Alzheimer's disease and controls, but amylin-containing pericytes displayed nuclear changes associated with cell death and reduced expression of the pericyte marker neuron-glial antigen 2. The impact of amylin on pericyte viability was further demonstrated in in vitro studies, which showed that pericyte death increased in presence of fibril- and oligomer amylin. Furthermore, oligomer amylin increased caspase 3/7 activity, reduced lysate neuron-glial antigen 2 levels and impaired autophagy. Our findings contribute to increased understanding of how aggregated amylin affects brain vasculature and highlight amylin as a potential factor involved in microvascular pathology in dementia progression.