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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., (Copyright © 2020. Published by Elsevier Inc.)

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.

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.

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2019 Wiley Periodicals, Inc.)

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.

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-β (TGF-β) 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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (© 2018 by The American Society of Hematology.)

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.

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.

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.

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 (Cav1.2 KO ). 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, Cav1.2 KO 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 Cav1.2 KO 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 Cav1.2 KO OPCs were identified by a Cre reporter, we establish that Cav1.2 KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca 2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca 2+ 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 Ca 2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca 2+ 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 Ca 2+ 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., (Copyright © 2017 the authors 0270-6474/17/3710038-14$15.00/0.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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, a pancreatic β-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.

As a transplant surgeon, my interest in glycobiology began through my research into ABO-incompatible allotransplantation, and grew when my goal became overcoming the shortage of organs from deceased human donors by the transplantation of pig organs into patients with terminal organ failure (xenotransplantation/cross-species transplantation). The major target for human "natural" (preformed) anti-pig antibodies is galactose-α(1,3)-galactose (the "Gal" epitope), which is expressed on many pig cells, including the vascular endothelium. The binding of human IgM and IgG antibodies to Gal antigens initiates the process of hyperacute rejection, resulting in destruction of the pig graft within minutes or hours. This major barrier has been overcome by the production of pigs in which the gene for the enzyme α(1,3)-galactosyltransferase (GT) has been deleted by genetic engineering, resulting in GT knockout (GTKO) pigs. The two other known carbohydrate antigenic targets on pig cells for human anti-pig antibodies are (i) the product of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene, i.e., N-glycolylneuraminic acid, and (ii) the product of the β1,4 N-acetylgalactosaminyltransferase gene, i.e., the Sd(a) antigen. Expression of these two has also been deleted in pigs. These genetic manipulations, together with others directed to overcoming primate complement and coagulation activation (the latter of which also relates to glycobiology) have contributed to the prolongation of pig graft survival in nonhuman primate recipients to many months rather than a few minutes. Clinical trials of the transplantation of pig cells are already underway and transplantation of pig organs may be expected within the relatively near future., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4+ and CD8+ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8+ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies.

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

Further attenuated measles vaccines were developed more than 50 years ago and have been used throughout the world. Recombinant measles vaccine candidates have been developed and express several heterologous virus protective antigens. Immunogenicity and protective actions were confirmed using experimental animals: transgenic mice, cotton rats, and primates. The recent development of measles vaccine-based vectored vaccine candidates has been reviewed and some information on recombinant measles vaccines expressing respiratory syncytial virus proteins has been shown and discussed., (Copyright © 2015. Published by Elsevier Ltd.)

The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Taenia solium cysticercosis is a major parasitic disease that affects the human health and the economy in underdeveloped countries. Porcine cysticercosis, an obligatory stage in the parasite life cycle, is a suitable target for vaccination. While several recombinant and synthetic antigens proved to be effective as vaccines, the cost and logistic difficulties have prevented their massive use. Taking this into account, a novel strategy for developing a multi-epitope low-cost vaccine is herein explored. The S3Pvac vaccine components (KETc1, KETc12, KETc7, and GK1 [KETc7]) and the protective HP6/TSOL18 antigen were expressed in a Helios2A polyprotein system, based on the 'ribosomal skip' mechanism mediated by the 2A sequence (LLNFDLLKLAGDVESNPG-P) derived from the Foot-and-mouth disease virus, which induces self-cleavage events at a translational level. This protein arrangement was expressed in transgenic tobacco cells. The inserted sequence and its transcript were detected in several Helios2A lines, with some lines showing recombinant protein accumulation levels up to 1.3 µg/g of fresh weight in leaf tissues. The plant-derived Helios2A vaccine was recognized by antibodies in the cerebral spinal fluid from neurocysticercosis patients and elicited specific antibodies in BALB/c immunized mice. These evidences point to the Helios2A polyprotein as a promising system for expressing multiple antigens of interest for vaccination and diagnosis in one single construction.

We hypothesized that pathological changes associated with elevations in annexin V-positive microparticles (MPs) following high-pressure exposures can be abrogated by ascorbic acid in a murine model. Mice exposed for 2 h to 790-kPa air and killed at 2 or 13 h postdecompression exhibited over threefold elevations in circulating MPs, as well as subgroups bearing Ly6G, CD41, Ter119, CD31, and CD142 surface proteins. There was evidence of significant neutrophil activation, platelet-neutrophil interactions, and vascular injury to brain, omentum, psoas, and skeletal muscles assessed as leakage of high-molecular-weight dextran. Prophylactic ascorbic acid (500 mg/kg ip) administration prevented all postdecompression neutrophil changes and vascular injuries. Ascorbic acid administration immediately after decompression abrogated most changes, but evidence of vascular leakage in the brain and skeletal muscle at 13 h postdecompression persisted. No significant elevations in these parameters occurred after injection of ascorbic acid alone. The findings support the idea that MP production occurring with exposures to elevated gas pressure is an oxidative stress response and that antioxidants may offer protection from pathological effects associated with decompression., (Copyright © 2015 the American Physiological Society.)

Central nervous system (CNS) injuries cause to variable disorders in people around the world without any decisive treatment. Use of embryonic stem cells (ESCs) would be helpful in repairing of neural system damages. Somatic cell nuclear transfer (SCNT) is a way for implanting ESCs with lowest possible rejection. In the present study, mouse nuclear transfers ESCs (ntESCs) ability in differentiation to oligodendrocyte progenitor cells (OPCs) was investigated by morphological study, RT—PCR and flow cytometry analysis. Bi—polar and tri—polar, OPCs were observed in stem cells cultured in differentiation medium after four weeks. Result of gene expression analysis demonstrated that differentiated stem cells were expressed most important OPCs related gene markers after differentiation period. Moreover, flow cytometry analysis carried out to confirm other results that showed differentiated stem cells significantly expressed NG2 and O4 as critical OPC surface markers. Taken together, it seems that mouse ntESCs showed highly potential for OPC differentiation and could be an appropriate candidate for stem cell therapies.

Vaccination involves inoculation of a subject with a disabled disease-causing microbe or parts thereof. While vaccination has been highly successful, we still lack sufficiently effective vaccines for important infectious diseases. We propose that a more complete immune response than that elicited from a vaccine may be obtained from immunization with a disease-causing virus modified to subject replication-essential genes to the control of a gene switch activated by non-lethal heat in the presence of a drug-like compound. Upon inoculation, strictly localized replication of the virus would be triggered by a heat dose administered to the inoculation site. Activated virus would transiently replicate with an efficiency approaching that of the disease-causing virus and express all viral antigens. It may also vector heterologous antigens or control co-infecting microbes.

In recent years, there has been an increased interest of researchers in developing efficient plant heterologous expression systems of proteins for a wide range of applications. It represents an alternative to the traditional strategy utilizing bacterial, yeast, insect or mammalian cells. New techniques of identification and characterization and effective methods of plant genetic transformation allow the range of recombinant protein products to be expanded. Great expectations are associated with the use of plants as bioreactors for the production of specific proteins of therapeutic interest. This strategy offers a number of advantages, the most important being: the possibility of a significant reduction in production costs, the safety of the products obtained and full eukaryotic post-translational modifications of proteins. A group of proteins of special interest is pharmaceuticals, and a number of successful experiments have confirmed the possibility of obtaining heterogeneous proteins with therapeutic potential: monoclonal antibodies, vaccine antigens, and a variety of cytokines. This work is focused on selected recombinant proteins belonging to those groups expression of which was achieved in plant cells. These proteins may be used in the future for therapy or prevention of viral, bacterial or cancer diseases.

Objectives: Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host., Key Findings: The yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles., Summary: Advances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development., (© 2014 Royal Pharmaceutical Society.)

There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: An increasing number of evidence suggests that pancreatic cancer contains cancer stem cells (CSCs), which may be relevant to the resistance of chemotherapy. Latexin (Lxn) is a negative regulator of stem cell proliferation and we investigate the effects of Lxn on CD133+ pancreatic cancer stem-like cells., Methods: CD133+ miapaca-2 cells, a human pancreatic carcinoma cell line, were isolated and sorted by magnetic activated cell sorting and flow cytometry. The capacity for self-renewal, proliferation, and tumorigenicity of CD133+ miapaca-2 cells was determined by the floating spheres test and tumor xenograft assays. Protein and mRNA expression of Lxn in CD133+ and CD133- miapaca-2 cells were detected by Western blotting and qRT-PCR, respectively. After CD133+ miapaca-2 cells were treated with Lxn in serum-free medium (SFM), cell proliferation was assayed with a Cell Counting Kit 8 (CCK-8) and apoptosis was analyzed by flow cytometry. The protein and mRNA expression levels of Bcl-2, bax, and c-myc were also analyzed., Results: We successfully isolated CD133+ miapaca-2 cells that exhibited the capacity for self-renewal in SFM, a proliferation potential in DMEM supplemented with FBS, and high tumorigenicity in nude mice. Lxn protein and mRNA expression levels in CD133+ miapaca-2 cells were significantly lower than those in CD133- cells. Lxn-treated CD133+ miapaca-2 cells exhibited increased apoptosis and low proliferation activity, down-regulation of Bcl-2 and c-myc expression, and up-regulation of Bax expression in a dose-dependent manner., Conclusions: Lxn induces apoptosis and inhibits the proliferation of CD133+ miapaca-2 cells. These changes are associated with down-regulation of Bcl-2 and c-myc and up-regulation of Bax.

NG2 (nerve/glia antigen-2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2-expressing (NG2(+) ) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2(+) cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter(+) astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen-sensitive and NG2 gene locus-dependent gene recombination could be detected in a small, but persistent population of cortical NeuN(+) neurons starting from the second postnatal week., (Copyright © 2014 Wiley Periodicals, Inc.)

Psoriasis is a chronic inflammatory skin disease with unknown aetiology. Infiltration of inflammatory cells as the initial event in the development of new psoriatic plaques together with the defined inflamed areas of such lesions argues for an immunological disease with a local production of a causal antigen. The auto-antigen Pso p27 is a protein expressed in the skin lesions. We recently demonstrated that Pso p27 is homologous to the core amino acid sequences of squamous cell carcinoma antigens 1 and 2 (SCCA1/2) and it is apparently generated from SCCA molecules by digestion with highly specific endoproteases. In this communication we demonstrate the generation of Pso p27 from SCCA1 with extracts from psoriatic scale and even more remarkably, the generation of Pso p27 from SCCA1 in the presence of mast cell associated chymase. These findings open up for new therapeutic strategies in psoriasis and probably also in other autoimmune diseases as Pso p27 epitopes have been detected in diseased tissues from patients with various chronic inflammatory diseases., (Copyright © 2014. Published by Elsevier B.V.)

The aim of the study was to detect the neocortical columns in the S1 field on frontal sections of brain of albino rats using the method of immunohistochemistry and the antibodies against neuronal (synaptophysin, neurofilament) and gliocyte (glial fibrillary acidic protein--GFAP, myelin basic protein) proteins. The examination of the expression of the major neurospecific antigens revealed that on thin sections (4 micromin) a column could be identified due to accumulations of the astrocytes and neuronal processes--axons and dendrites. GFAP expression study also showed that cortical layer I usually contained multiple large astrocytes with branching processes, as well as numerous smaller processes with high intensity of expression. Synaptophysin content was high in all the layers of the cortex, but the most intense reaction was detected in the molecular layer, similarly with the intensity of GFAP reaction. The expression of myelin basic protein was detected in accordance with the radially extending myelinated processes of the neurons in the cortex.

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73  μ g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25  μ g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

The immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. Characterized attenuated alphavirus mutants contain changes to the genomic 5' UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction. In an attempt to create more potent replicon vaccines, we engineered a panel of Venezuelan equine encephalitis-Sindbis virus chimeric replicons that contained these attenuating mutations. Modified replicons were ranked for antigen expression and IFN induction levels in cell culture and then evaluated in mice. The results of these studies showed that differences in antigen production and IFN induction in vitro did not correlate with large changes in immunogenicity in vivo. These findings indicate that the complex interactions between innate immune response and the replicon's ability to express antigen complicate rational design of more potent alphavirus replicons., (© 2013 Elsevier Inc. All rights reserved.)

The evolution in proteomics approaches is notable, including quantitative proteomics and strategies for elucidation of post-translational modifications. Faster and more accurate mass spectrometers as well as cleverer bioinformatics tolls are making the difference in such advancement. Among the wide range of research in plant proteomics, biopharmaceutical production using plants as "biofactories" and the screening of new activities of new molecules, in this case, peptides, are quite important regarding translational proteomics. The present review is focused on "recombinant proteins and bioactive peptides", with biopharmaceuticals and cyclotides chosen as examples. Their application and challenges are focused on a "translational proteomics" point of view, in order to exemplify some new areas of research based on proteomics strategies. This article is part of a Special Issue entitled: Translational Plant Proteomics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT)., Methods and Results: Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast., Discussion and Conclusion: This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-l-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to l-amino acids but not to recognize the corresponding d-enantiomers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Modulation of cell surface properties via functional modification is of great interest in cell-based therapies, drug delivery, and in transfusion. We study the in vivo circulation, immuogenicity, and mechanism of clearance of hyperbranched polyglycerol (HPG)-modified red blood cells (RBCs) as a function of molecular properties of HPGs. The circulation half-life of modified cells can be modulated by controlling the polymer graft concentration on RBCs; low graft concentrations (0.25 and 0.5 mM) showed normal circulation as that of control RBCs. Molecular weight of HPG did not affect the circulation of modified RBCs. HPG grafting on RBCs reduced CD47 self-protein accessibility in a graft concentration-dependent fashion. HPG-grafted RBCs are not immunogenic, as is evident from their similar circulation profile upon repeated administration in mice and monitoring over 100 days. Histological examination of the spleen, liver, and kidneys of the mice injected with modified RBCs revealed distinct differences, such as elevated iron deposits and an increase in the number of CD45 expressing cells at high graft concentration of HPGs (1.5 mM); no changes were seen at low graft concentration. The absence of iron deposits in the white pulp region of the spleen and its presence in the red pulp region indicates that the clearance of functional RBCs occurs in the venous sinuses mechanical filtering system, similar to the clearance of unmodified senescent RBCs. HPG modification at grafting concentrations that yield long circulation in mice produced camouflage of a large number of minor blood group antigens on human RBCs, demonstrating its utility in chronic transfusion. The normal circulation, nonimmunogenic nature, and the potential to modulate the circulation time of modified cells without toxicity make this HPG-based cell surface modification approach attractive for drug delivery and other cell-based therapies.

This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G-) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4(+) T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G- bacteria and may be relevant for the use of bacterial carriers in vaccine development.

Objective: To co-express antigen and antibody in E.coli periplasm in which the antigen and antibody will fold correctly and bind to each other., Methods: Human IL-1β gene and mouse anti-human IL-1β single chain variable fragment (anti-hIL-1β scFv) were inserted into the down stream from the NlpA and pelB leading peptides of pBFD vector respectively. The recombinant plasmid was named pBFD-Ab-Ag. The E.coli DH5α transformed with pBFD-Ab-Ag was induced by IPTG to co-express antigen and antibody. After the outer membrane of E.coli DH5α was broken (spheroplast formation), the bacteria were incubated with mouse anti-hIL-1β antibody labeled by FITC. The co-expression and interaction of antigen and antibody was analyzed by flow cytometry (FCM) based on fluorescence signal intensity., Results: The FCM detection showed the strong signal in E.coli DH5α transformed with pBFD-Ab-Ag and the positive rate was significantly improved., Conclusion: The results showed that the antigen and antibody can fold and bind to each other efficiently in E.coli periplasm. The system can make antibody screening process easier and also provide a novel method for protein-protein interaction.

The autoimmune regulator is a critical transcription factor for generating central tolerance in the thymus. Recent studies have revealed how the autoimmune regulator targets many otherwise tissue-restricted Ag genes to enable negative selection of autoreactive T cells.

Restenosis is a major complication of coronary angioplasty, at least partly due to the fact that the origin and identity of contributing cell types are not well understood. In this study, we have investigated whether pericyte-like cells or mesenchymal stem cells (MSCs) from the adventitia contribute to restenosis. We demonstrate that while cells expressing the pericyte markers NG2, platelet-derived growth factor receptor β, and CD146 are rare in the adventitia of uninjured mouse femoral arteries, following injury their numbers strongly increase. Some of these adventitial pericyte-like cells acquire a more MSC-like phenotype (CD90+ and CD29+ are up-regulated) and also appear in the restenotic neointima. Via bone marrow transplantation and ex vivo artery culture approaches, we demonstrate that the pericyte-like MSCs of the injured femoral artery are not derived from the bone marrow, but originate in the adventitia itself mainly via the proliferation of resident pericyte-like cells. In summary, we have identified a population of resident adventitial pericyte-like cells or MSCs that contribute to restenosis following arterial injury. These cells are different from myofibroblasts, smooth muscle cells, and other progenitor populations that have been shown to participate in the restenotic process., (Copyright © 2012 S. Karger AG, Basel.)

The import of functional nucleic acids like messenger RNA into mammalian cells has proven to be a powerful tool in cell biology and several delivery systems have been described. However, as targeting of particular cell types is a major challenge and RNA vaccination represents a promising means for the induction of cellular immune responses, there is a need for novel delivery systems that permit the introduction of functional messenger RNA to the cytosol of immune cells. Here, we describe a delivery system based on the yeast Saccharomyces cerevisiae that allows the delivery of functional messenger RNA to mammalian antigen-presenting cells such as human dendritic cells. Further, we present a method to prove antigen processing and presentation by stimulation of human autologous T lymphocytes.

Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan., (Copyright © 2012 Elsevier Inc. All rights reserved.)