Kathleen L. Hefferon

Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins

Transgenic plants present enormous potential as a cost-effective and safe platform for large-scale production of vaccines and other therapeutic proteins. A number of different technologies are under development for the production of pharmaceutical proteins from plant tissues. One method used to express high levels of protein in plants involves the employment of plant virus expression vectors. Plant virus vectors have been designed to carry vaccine epitopes as well as full therapeutic proteins such as monoclonal antibodies in plant tissue both safely and effectively. Biopharmaceuticals such as these offer enormous potential on many levels, from providing relief to those who have little access to modern medicine, to playing an active role in the battle against cancer. This review describes the current design and status of plant virus expression vectors used as production platforms for biopharmaceutical proteins.

Nutritionally Enhanced Food Crops; Progress and Perspectives

Great progress has been made over the past decade with respect to the application of biotechnology to generate nutritionally improved food crops. Biofortified staple crops such as rice, maize and wheat harboring essential micronutrients to benefit the world’s poor are under development as well as new varieties of crops which have the ability to combat chronic disease. This review discusses the improvement of the nutritional status of crops to make a positive impact on global human health. Several examples of nutritionally enhanced crops which have been developed using biotechnological approaches will be discussed. These range from biofortified crops to crops with novel abilities to fight disease. The review concludes with a discussion of hurdles faced with respect to public perception, as well as directions of future research and development for nutritionally enhanced food crops.

Plant Virus Expression Vector Development: New Perspectives

Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants.

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Abstract Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVYo and PVYN under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations.

Expression and Mutational Analysis of Autographa californica Nucleopolyhedrovirus HCF-1: Functional Requirements for Cysteine Residues

ABSTRACT The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

Bioengineering Hairy Roots: Phytoremediation, Secondary Metabolism, Molecular Pharming, Plant-Plant Interactions and Biofuels

Hairy root cultures are an efficient tool to understand plant biology, biotechnology and other applied sciences. In particular such cultures have revealed many cues of plant cells related to growth, development, physiological and biochemical processes. Thus, hairy roots are used to study secondary metabolism and production of bioactive compounds such as flavonoids. Transgenic roots are used as biofoactories to produce heterologous recombinant proteins for pharmaceutical purposes. Furthermore, they have shown promising applications in phytoremediation and restoring the environment because of advantages in the reduction of toxic organic and inorganic pollutants from soil, air, wastewater, groundwater and biowaste. This review focuses on the recent progress of bioengineering hairy root culture systems.

Plant virus expression vectors for biopharmaceutical production

C nanotubes have been studied for a wide variety of applications included in medicine as drug delivery systems, targetable materials and diagnostic tools. Carbon nanotubes, toxic in their pristine forms, have been functionalized with many different techniques to make them biocompatible. It has been showed the potential use of well functionalized carbon nanotubes (f-CNTs) as ultrasound contrast agents. The interaction of f-CNTs and primary immune cells has been studied and, f-CNTs have been proposed as immunomodulators showing their potential as activators of the immune systems. Moreover, it was recently evaluated the possibility of taking advantage of immunostimolatory properties of f-CNTs against microgravity immune function dysregulation. The results proved that the capacity of f-CNTs to stimulate immune cells have very interesting broad future applications not only in immunotherapy or as vaccine adjuvants, as recently suggested, but also to contrast spaceflight immune cells functionality suppression. Results from different investigations, functionality assays and their potential as theranostic materials will be presented and discussed.T idiopathic inflammatory myopathies (IIM) are systemic autoimmune disease, which is caused by an immune-mediated inflammation and characterised with proximal muscle weakness. Myositis-specific auto-antibodies (MSAs) are associated with the disease and can be detected in patients’ sera. These MSAs determine subgroups which are different in symptoms, severity, prognosis and genetic background. During the recent development in knowledge about auto-antibodies the roles of MSAs in diagnosis and prognosis have changed. Anti-TIF1y autoantibody is presented as a tumor-specific MSA led to a 155-kDa/140-kDa protein complex and also showed an association with serious juvenile and adult DM. The association of malignant diseases with myositis is also well known. Risk for tumor in dermatomyositis (DM) is 3-fold, and 1.3-fold in (PM). Based on these data, searching malignancy is one of the most important steps after myositis diagnosis. During this research our aim was to determine the clinical characteristics associated with the presence of anti-TIF1y antibodies and to define the serological subgroup in cancer-associated (CAM) myositis, particularly the tumor specificity and of the mentioned antibody. Patients were cared by the Division of Clinical Immunology, Institute of Internal Medicine and University of Debrecen. We examined 202 patients with IIM. Inclusion criteria was the presence of finished antibody testing, 12 cases showed positivity in anti-TIF1y. One of these patients had CAM. We examined the differences between the anti-TIF1y positive (n=12) and negative CAM (n=51) groups concerning symptoms, lab values, cancer type. The anti-TIF1y antibody positivity has been presented in 5.9% of our patients and associated with severe skin symptoms. Although earlier studies claimed that CAM patients are similarly characterized by severe skin symptoms, in our cohort respectively prevalence of Gottron’s sign and Heliotrop rash were significantly higher in anti-TIF1y positive cases. This research could not prove the tumor-specificity of anti anti-TIF1y antibody, but our results confirm that the presence of this antibody separate a subgroup of myositis different in clinical symptoms and severity.Background: The gastrointestinal (GI) tract is a major site of viral replication, CD4+ T cell depletion, viral dissemination and reservoir formation. Chronic GI inflammation, a hallmark of progressive HIV/SIV infection is associated with disruption of the intestinal epithelial barrier leading to microbial translocation and contributing to localized and systemic immune activation/ inflammation which drives AIDS disease progression. Recent evidence suggests critical roles for small non-coding regulatory RNA molecules called microRNAs (miRNAs)that regulate gene expression through post-transcriptional gene silencing in controlling and managing certain aspects of the inflammatory process.P (PON) is a potent oxidizing and nitrating agent with a biological half-life of approximately 10 ms. It is produced in vivo by diffusion-controlled reaction of nitric oxide and superoxide anion. It can oxidize and/or nitrate many amino acids causing changes in protein structure and function. The changes may lead to the pathogenesis of several inflammatory diseases including Rheumatoid Arthritis (RA). In this study, IgG was isolated from healthy subjects’ sera on protein A-agarose affinity column and PON was synthesized by rapid quenched flow method. PON-modified IgG (PON-IgG) was prepared by incubating IgG with PON at 37oC for 30 min and maintaining pH at 10-11. Physicochemical alterations in PON-modified IgG were monitored by UV, fluorescence, CD and FT-IR spectroscopy, and SDS-PAGE. Oxidation and aggregation were assessed as free thiols, protein carbonyls, and thioflavin T and congo red binding. Nitrotyrosine, dityrosine and nitrotryptophan were also quantified. Formation of 3-nitrotyrosine was verified by LC-MS and HPLC, and attachment of PON to IgG was elucidated by MALDI-TOF mass spectrometry. PON-modified IgG exhibited hyperchromicity at 278 nm along with bathochromic shift and appearance of a new peak at 420 nm, decrease of tryptophan and tyrosine fluorescence, loss in β-sheet and appearance of new peak in FT-IR, compared to native IgG. SDS-PAGE results revealed concentration dependent decrease in the band intensity of PON-IgG compared to native IgG. Experimentally induced antibodies against PON-IgG showed high titre antibodies and specific binding may be due to generation of highly immunogenic neo-epitopes on PON-IgG. PON-IgG binding with circulating autoantibodies of RA patients were compared with the experimentally induced antibodies against PON-IgG and its significance was analysed using biostatistical tools. The results so far suggest likely involvement of PON-IgG as an autoantigen in the induction and/or progression of RA.Introduction: bezoars are masses formed by the accumulation of intraluminal nondigestible substances that can lead to obstruction of the stomach and the small intestine. The anatomical changes in the gastrointestinal tract are known to cause bezoar formation. The so-called Rapunzel syndrome is the extension of the bezoars down to the duodenum and the jejunum, which is a rare condition. This may occur in subjects with mental retardation and/or psychiatric disorders. Case presentation: we present a 7-year-old female with primary biliary cirrhosis who was admitted to our department with hematemesis, loss of weight and fatigue complaints along the last 6 months, in whom a giant trichobezoar was identified through endoscopy which completely filled the stomach and duodenum causing partial obstruction and needed surgical interference for removal and psychiatric therapy. We believe that -after MEDLINE search along the last 10 years- this is the first Egyptian case presentation of Rapunzel syndrome in a child with autoimmune liver disease (primary biliary cirrhosis) and trichotillomania presented at very young age. The link between autoimmune liver diseases and neuropsychiatry disorders is still poorly understood and this case report may help us to understand the link between the two disorders.Conclusions: trichobezoars should be considered as a differential diagnosis in children complaining of recurrent abdominal pain with epigastric mass and progressive weight loss. The link between autoimmune liver diseases and neuropsychiatry disorders is still poorly understood and this case may help us to understand the link between the two disorders. It is an original case report of interest to pediatricians especially gastroenterology, hepatology and neuropsychiatry specialists.T objective of this study is to deliver anti-pPKCθ (T538) into T cells (hPBMCs) by using cell penetrating peptide mimics (CPPMs) to neutralize PKCθ activity both in vitro and in vivo, with the eventual goal of treating aplastic anemia (AA). AA is an immune-mediated bone marrow failure disease caused by T helper type 1 (Th1) autoimmune responses, which destroy blood cell progenitors. It was previously reported that protein kinase C theta (PKCθ), expressed specifically in T cells, plays an important role in T cell signaling by mediating Th1 differentiation. Mice treated with Rottlerin, a pharmacological inhibitor of PKCθ, were rescued from the disease when PKCθ phosphorylation was inhibited. Furthermore, humanized antibodies are increasingly gaining attention as therapies. The delivery of antibodies could be achieved via cell penetrating peptides (CPPs), which are able to internalize cargo into cells. Here, we designed, synthesized and characterized CPPMs to increase delivery efficiency of an antibody against phosphorylated PKCθ (T538), which subsequently interfered with the function of the kinase. We designed an in vitro delivery method for the CPPM/anti-pPKCθ complex then assessed T cell activation and AA disease marker expression. Also, we generated an in vivo humanized mouse model of AA and tested the complex for delivery and effect on survival of these mice. Altogether the results reveal that PKCθ may be an optimal target for bone marrow failure treatment and intracellular antibody delivery may represent a novel approach for AA treatment.Material and Methods: Our study is a comparative clinical research, done in the University Clinical Center in Prishtina and in cooperation with specialized allergologic center Ylli in Prishtina. The including criteria was: 60 adult patients diagnosed with allergic asthma Intermitent mild, Mild persistent, Moderate persistent Asthma (according to GINA) aged between 15 and 30 years, both sexes. 30 patients are treated with specific Immunotherapy (SCIT) and 30 of them with another anti-asthmatic drugs.V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed.S coating tumor cells with foreign antigenic peptide may render coated tumor susceptible to immune recognition and elimination, thereby bypassing immune tolerance. Here we create a therapeutic chimeric protein comprised of a tumor-homing module fused to a functional cargo domain containing foreign antigenic peptide. The tumor-homing module is comprised of mesothelin-specific single chain variable fragment that specifically binds to mesothelin, commonly overexpressed in ovarian tumors. The functional cargo domain is comprised of Fc (IgG2a) protein and MHC class I-restricted foreign CD8+ T cell epitope flanked by furin cleavage sites, which can be recognized by furin highly expressed in the tumor microenvironment. We show that our therapeutic protein specifically loaded antigenic epitope onto MHC class I of bound tumor cells, rendering them susceptible to antigen-specific CD8+ T cell-mediated killing for potent antitumor effects in vitro and in vivo. Our findings have important implications for bypassing immune tolerance to enhance cancer immunotherapy.C inflammation plays a role in breast carcinogenesis. Local inflammation would be manifested by increased expression of pro-inflammatory markers [interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α)] to anti-inflammatory marker [transforming growth factor-β (TGF-β)] in breast tissue. It was assessed if local inflammatory state in breast tissue would affect the mammographic density, a breast cancer risk factor, among 82 premenopausal and 82 postmenopausal breast cancer patients. For each patient, six cores of normal breast tissue, located at >1.0 cm from the tumor, were extracted from mastectomy blocks and used to build up tissue microarray (TMA) blocks. TMA cut sections were stained by immunohistochemistry and digitized. The protein expression levels of inflammatory markers were visually estimated (intensity and proportion of positive cells) in normal epithelium present on digitized stained TMA sections. Quick score was obtained by multiplying the intensity by the proportion of positive cells. The proto anti-inflammatory marker expression ratios (IL-6/TGF-β and TNF-α/TGF-β) were calculated in three categories based on the quick score; anti-inflammatory (pro-inflammatory marker<anti-inflammatory marker), neutral (pro-inflammatory marker=anti-inflammatory marker) or pro-inflammatory state (pro-inflammatory marker>anti-inflammatory marker). Mammographic density was evaluated by a computer-assisted method. Associations were assessed by multivariate generalized linear models. Pro-inflammatory state of TNF-α/TGF-β among premenopausal women and IL-6/TGF-β among postmenopausal women were associated with higher percent mammographic density compared to the anti-inflammatory or the neutral state (p=0.014 and p=0.005, respectively). Affecting the expression of inflammatory markers in breast tissue may provide attractive targets for future breast cancer preventive strategies.S germinal center (Spt-GC) B cells and follicular helper T cells (Tfh) generate high affinity autoantibodies involved in the development of systemic lupus erythematosus (SLE). Toll like receptors (TLRs) play a pivotal role in SLE pathogenesis. While previous studies have focused on the B cell intrinsic role of TLR-MyD88 signaling on immune activation, autoantibody repertoire and systemic inflammation, a thorough investigation of the mechanisms by which TLRs control the formation of Spt-GCs remains unclear. Using non-autoimmune C57BL/6 (B6) mice deficient in MyD88, TLR2, 3, 4, 7 or 9, we identified B cell-intrinsic TLR7 signaling as a prerequisite to Spt-GC formation without the confounding effects of autoimmune susceptibility genes and the overexpression of TLRs. TLR7 deficiency also rendered autoimmune B6. Sle1b mice unable to form Spt-GCs, leading to markedly decreased autoantibodies. Conversely, B6.yaa and B6.Sle1b.yaa mice expressing an extra copy of TLR7 and B6.Sle1b mice treated with aTLR7 agonist had increased Spt-GCs and Tfh. Further, TLR7/ MyD88 deficiency led to compromised B cell proliferation and survival after B cell stimulation both in vitro and in vivo. In contrast, TLR9 inhibited Spt-GC development. Our findings demonstrate an absolute requirement of TLR7 and anegative regulatory function for TLR9 in Spt-GC formation under non-autoimmune and autoimmune conditions. Our data suggest that, under non-autoimmune conditions, Spt-GCs initiated by TLR7 produce protective antibodies. However, in the presence of autoimmune susceptibility genes TLR7 dependent Spt-GCs produce pathogenic autoantibodies. Thus, a single copy of TLR7 in B cells is the minimal requirement for breaking the GC-tolerance checkpoint.E research in a relatively young scientific discipline called photo immunology is constantly revealing new insights into the immunomodulatory effects of ultraviolet radiation (UVR) from sunlight and of vitamin D. Evidence from up to date studies suggests that exposure to UVR and vitamin D deficiency are associated with immune suppression and an increased susceptibility to infection, along with a diminished reaction to some types of vaccinations. Ultraviolet radiation is known to adversely affect the immune system, hence its use in the context of an overactive immune system (e.g. autoimmune disease) via immunosuppression. Along with its harmful side effects, UVR is likely to lead to an increased vitamin D status. Vitamin D is fundamental for a positive immunomodulatory function that, probably, outweighs the negative effect of UVR, which is limited mainly to the approximately 48 hours post exposure and can be controlled with proper precautions. Recent studies have demonstrated that vitamin D deficiency is related to infection severity, including increased length of hospitalization stay and increased mortality in those admitted to intensive care units. Conversely, no consistent protective properties of vitamin D supplementation were evident in prospective clinical trials, except for vitamin D deficient individuals. Thus far, dietary vitamin D supplements have not been found to enhance the immune response to vaccines, and the inoculation rate is not determined by vitamin D status. The myriad of mechanisms that mediate UVR induced immune-modulation is complex and controversial. The suppressive and the stimulatory influences of UV radiation and vitamin D on the immune system, conjoint or independent, are key factors in achieving optimal immunogenicity. This issue is of special concern given the constantly increasing amount of radiation in our atmosphere. Prospective public health implications regarding routine vaccination scheduling and policies; as well as sunscreen modalities for preventing UVR-induced damage are further discussed.Antitumor effect of coriander honey (500 mg/kg) was investigated in mature mice bearing Ehrlich carcinoma with special reference to immune status. The coriander honey showed decrease in tumor volume, packed cell volume and viable cell count, and increased the non-viable cell count and mean survival time thereby increasing life span of EAC tumor bearing mice. The results of the effect of honey on immunological status in the mice bearing Ehrlich carcinoma observed that the immunoglobulin levels (M, G and A) were raised in all groups if compared with the control group all over the experimental period. The rises were differed according to the treatment. It was clear that the phagocytic activity in mice bearing carcinoma was reduced if compared with the control group while it increased in the treated group with honey. There was reduction in the stimulation indices of stimulation indices of lymphocyte transformation of mice bearing Ehrlich carcinoma. Delayed hypersensitivity skin test revealed that the Ehrlich carcinoma reduced the reaction especially after 72 hours post inoculation with bovine serum albumin. The reaction in the Ehrlich carcinoma and subsequently treated with honey showed a rise in thickness ranging from 0.62 mm skin thickness in case of Ascites where the solid state was more higher 0.73 mm thickness. It could be concluded that, the coriander honey exhibited antitumor effect by increases cell mediated and immunoglobulin levels, in EAC bearing mice.S lupus erythematosus is an autoimmune disease characterized by elevated production of auto-reactive antibodies and systemic inflammation. Nine out of ten patients suffering from SLE are female. Sex hormones have been extensively studied for their potential effects on disease pathogenesis. It is thus known that testosterone protects while estrogens exacerbate disease development. We have recently described a population of Gr1+CD11b+ cells that are upregulated in male lupusprone (NZB x NZW)F1 mice. These cells are driven by testosterone in a dose-dependent manner, as revealed by castration and hormonal reconstitution studies. Interestingly, although Gr1+ cells from both male and female prepubescent (NZB x NZW)F1 mice are immunosuppressive, the suppressive capacity is driven by different mechanisms and only male-derived cells remain immunosuppressive after puberty. In support hereof, adult 9 week old male mice depleted of Gr1-expressing cells develop increased levels of antibodies to both thymus-dependent antigens (NP-CGG) and nuclear auto-antigens, while a similar depletion strategy in age-matched females had no effect on either NP-specific antibody production or the spontaneous appearance of anti-nuclear autoantibodies. Further studies revealed that Gr1+ cell depletion in adult (NZB x NZW)F1 male mice resulted in increased levels of TFH and GC B cells after immunization. We conclude that a population of testosteronedriven Gr1+ cells present in (NZB x NZW)F1 lupus-prone male mice exert a hitherto unappreciated role in controlling activation of the adaptive immune system and the development of lupus-like disease. Manipulation of these cells represents an interesting new target for immunotherapy in autoimmune disorders with a female predominance.H simplex virus type 1 (HSV-1) infection was examined on murine J774A.1 and RAW 264.7 macrophage cell lines unpolarized (M0) or polarized by IFN-γ and or cytokines to either an M1 (pro inflammatory) or M2 (immunomodulatory and tissue remodeling) phenotype. Dengue virus2 (DENV2) infection of RAW264.7 cells was also investigated. Morphology, cell viability, expression of cell surface markers, expression ratios of suppressor of cytokine signaling molecules SOCS1/ SOCS3, and capacity to replicate HSV-1 were determined. Morphological differences were abrogated by virus infection. Infection with either virus diminished expression of CD14 and CD86 in M1 populations, cells which exhibited significant decreases (p<0.001) in cell viability at 24 hours post infection. Western blotting suggested that SOCS1/SOCS3 expression ratios differed between uninfected and HSV-1-infected RAW264. 7 M1 cells, while ratios in the uninfected cells and the DENV2-infected M1 cells remained similar. Flow cytometry demonstrated that this ratio between uninfected M1 cells and HSV-1-infected M1 phenotypes fell from 6:1 to 1:1 in M1 phenotypes of J774A.1 cells. SOCS3 expression increased 7-fold in the M1 infected J774A.1 cells compared to uninfected M1 cells. Threeto four-fold decreases in HSV-1 yield occurred by 24 hours after infection in the M1 subpopulations. This decrease may reflect the anti-inflammatory effect of the relative increases in S0CS3 expression in these cells and/or the decreased survival of M1 cells after virus infection. Increases in SOCS3 expression by theHSV-1-infected M1 populations suggest a shift in the cell’s ability to respond to virus, a change not seen in DENV2 infection.L tumor antigen-specific T cell-NK cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of the T cell responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+ T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/and RAG-/-γc-/mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. In 2D and 3D co-culture systems, we observed that PMA/ionomycin-stimulated CD8+ T cells form multiple contacts with naive NK lymphocytes. Data show that NK cells interacting with activated CD8+ T cells show an up-regulation of CD25 and CD69 expression mediated by intercellular contacts, and activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, p38. On the other hand, interacting NK cells down-regulate CD25 molecule expression on CD8+ T cells and promote differentiation of central-memory CD44+CD62L+ T cells. CD8+ T cells display an elevation in the phosphorylation of Stat1 and down-regulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Moreover, significant changes in the cytosolic and mitochondrial Ca2+, production of mitochondrial ROS, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and non-protein thiols (mostly, reduced glutathion) were observed in a reciprocal T cell-NK cell interaction. These results highlight the importance of mitochondrial activity in the re-modeling of activation signaling and memory differentiation of interacting CD8 T cells and NK cells. These results will help refine cancer immunotherapeutic strategies.

Current Status of Plants as Vaccine Production Platforms

Plant-derived biopharmaceuticals offer enormous potential as a cost-effective, rapid and safe means to produce vaccine and therapeutic proteins. Plant-made vaccines can be administered orally to elicit a mucosal immune response, and represent a method by which vaccine coverage can be improved for many who reside in developing countries. Vaccines such as these offer great promise on many levels, from providing relief to those who have little access to modern medicine, to producing large-scale stockpiles of vaccines available to offset global pandemics, and even to playing an active role in the battle against cancer. Plant-derived vaccines can both deliver an antigen to the mucosal immune system in the form of a food product, as well as prevent the antigen from degradation as it passes through the gastrointestinal tract. Both transgenic plants and plant virus expression vectors are routinely used to express biopharmaceutical proteins. The following review details recent advances concerning the production of vaccines against Hepatitis B virus, Human papilloma virus, Influenza virus and Non Hodgkins Lymphoma using plant expression platforms.

Recent Patents Involving Virus Nucleotide Sequences; Host Defense, RNA Silencing and Expression Vector Strategies

Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.