title: Infiltration of immune T cells in the brain of mice with herpes simplex virus-induced encephalitis

text: Herpes simplex virus (HSV) infection of mice can induce viral encephalitis. Using two-fluorochrome immunofluorescence, our present study shows that though there is extensive myelin loss and necrosis in the brain stem of mice with HSV encephalitis, only some oligodendrocytes, astrocytes and microglial cells are infected. T cells that express CD4 or CD8 and a large number of CD4(+), F4/80(+) macrophages are present in perivascular infiltrates close to and in contact with HSV-infected cells in areas of massive myelin loss. These findings suggest that the resultant infiltration of immune cells into the brain during HSV-1 infection may cause as much damage as the virus itself.


title: Studies on the intrathecal humoral immune response in canine distemper encephalitis()

text: Albumin and IgG were quantitated in paired cerebrospinal fluid (CSF) and serum samples from dogs with demyelinating canine distemper virus (CDV) infection by means of rocket immunoelectrophoresis. The IgG index as indicator for intrathecal immunoglobulin synthesis was normal in animals with non-inflammatory demyelinating lesions and elevated in dogs with inflammatory myelin lesions. Specific antibodies against CDV and myelin were quantitated in CSF and serum from 8 dogs with an elevated IgG index. Eight of these dogs had significant amounts of antimyelin antibody and 4 dogs had neutralizing anti-CDV antibody in the CSF. Whereas the pathogenetic significance of antimyelin antibodies remains uncertain, the intrathecal antiviral immune response provides a plausible explanation for immunopathologic destruction of myelin in distemper.


title: Differential expression and regulation of major histocompatibility complex (MHC) products in neural and glial cells of the human fetal brain

text: The cells of the central nervous system (CNS) have the peculiarity of physiologically expressing very low levels of HLA molecules. In multiple sclerosis (MS), however, as in endocrine autoimmune diseases, there is a marked increase of HLA expression in the tissue (i.e. the plaques) and this is attributable not only to infiltrating cells but also to the astrocytes. To gain an insight into the regulation of HLA in the different cell types in the CNS and to compare it to that observed in the endocrine organs, we have studied the effect of the lympho/monokines interferon (IFN)-α and -γ, tumour necrosis factor (TNF)-α, and interleukin (IL)-2 and other agents on this aspect of the biology of human fetal brain cells in culture. A two-colour immunofluorescence technique which combines antibodies to diverse CNS cell markers and monoclonal antibodies (MoAbs) to the non-polymorphic region of HLA molecules was used throughout this study. In control cultures, only astrocytes expressed MHC class I, but after incubation with either IFN-γ or TNF-α oligodendrocytes acquired class I expression. Surprisingly, astrocytes became spontaneously class II positive in culture and this was greatly enhanced by IFN-γ. Other agents such as IL-2, epidermal growth factor, phorbolmyristate acetate and lectins had no effect. The expression of HLA molecules in the cells of the CNS both in basal conditions and in response to lymphokines is therefore selective and highly heterogenous, thus reflecting their intrinsic biological diversity. These findings may help to explain the features of the immunopathology of MS and also of latent viral infections of neural cells.


title: Comparative analysis of virus-specific antibodies and immunoglobulins in serum and cerebrospinal fluid of subacute measles virus-induced encephalomyelitis (SAME) in rats and subacute sclerosing panencephalitis (SSPE)

text: The intrathecal humoral immune response was analysed in patients with subacute sclerosing panencephalitis (SSPE) and Lewis rats with subacute measles virus (MV)-induced encephalomyelitis (SAME). SSPE patients as well as SAME rats revealed oligoclonal, intrathecal antibody synthesis with MV specificity. SAME rats synthesized MV-specific antibodies intracerebrally to a higher extent than SSPE patients. Although a restricted isoelectric pattern of MV-specific antibodies was detected in the cerebrospinal fluid (CSF) of SSPE patients as well as of SAME rats, the heterogeneity within clusters of immunoglobulin bands was higher in the rat specimens. Increase in the blood-brain barrier permeability for albumin was exclusively detected in SAME rats but not in SSPE patients. These data suggest that the rat model offers excellent opportunities to study the initial humoral events in MV-induced encephalitides.


title: Bone-marrow derived macrophages as targets for the replication of mouse hepatitis virus type 3

text: Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day 10 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.


title: Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system

text: Proliferation of central nervous system (CNS) glia in response to peripheral nerve injury occurs without apparent participation of cells of the immune system. It is shown here that following transection of the rat facial nerve there is strongly elevated expression of class I, and to a lesser extent, class II antigens of the major histocompatibility complex (MHC) in the facial nucleus. It is demonstrated by double-immunofluorescence studies that the cells responsible for increased levels of MHC class I antigens are endogenous brain microglia. These findings emphasize the thought that microglia are immunocompetent cells, but, at the same time, raise the possibility for a non-immubological function of MHC antigens under conditions of neural regeneration.


title: Microglial cell response to neuronal degeneration in the brain of brindled mouse

text: Reactive changes of microglia in response to neuronal degeneration were investigated in the brains of brindled mottled mice with immunocytochemical technique. This mutant has a genetic defect in copper metabolism and spontaneous neuronal degeneration develops around postnatal day 10, in particular in the parasagittal regions of the cerebral cortex and thalamus. The antibodies to macrophage specific antigen, F4/80 and to type-three complement receptor, Mac-1 were used for the study. Reactive morphological changes of microglia, which are immuno-reactive to the antibodies to F4/80 and/or Mac-1, were demonstrated in areas corresponding to those of neuronal degeneration, coincident with the emergence of cells expressing major histocompatibility complex class II, Ia, antigen. Some of the Ia expressing cells had morphological features of ramified microglia, while others were rod shaped with few processes and were mostly located in the perivascular regions. The focal nature of such cellular changes suggests that signal(s) from the degenerating neurons may be responsible for microglial activation and cellular expression of the Ia antigen in the brain of the brindled mouse.


Immunological activities of milk


title: Virus-specific and autoreactive T cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis()

text: Using a recently described technique for expanding of human T lymphocyte populations from cerebrospinal fluid (CSF), we investigated the local cellular immune response in a patient with chronic rubella panencephalitis. A total of 328 T cell lines (TCLs) was established by seeding CSF cells at limitin dilution into histoplates in the presence of irradiated feeder cells and phytohemagglutinin (PHA)-containing conditioned medium. 80% of TCLs expressed the CD4(+) CD8(−), 5% the CD4(−) CD8(+) phenotype and 15% of TCLs contained different proportions of CD4(+) and CD8(+) cells. Of 191 TCLs analyzed, 85 were cytotoxic, as shown by their lectin-dependent cytotoxicity against allogeneic uninfected target cells. Eight of them demonstrated specificity for the autologous, rubella virus-infected target cells. When testes for antigen-specific proliferative activity, 26 TCLs responded to rubella antigen, 16 TCLs reacted to myelin basic protein (MBP), four TCLs to proteolipid protein (PLP), four to galactocerebrosides and two to actin. Fourteen out of 16 MBP-specific TCLs also responded, to a minor degree, to rubella antigen and/or actin. The results showed that the persisting rubella infection had given rise to autoreactive T cells. Virus-induced autoreactivity to brain antigens may be an important pathogenic mechanism in other chronic inflammatory disorders of the CNS.


title: The tumor phenotype and the human gene map()

text: The tumor phenotype is associated with the rearrangement of genetic information and the altered expression of many gene products. In this review, genes associated with the tumor phenotype have been arranged on the human gene map and indicate the extent to which the tumor phenotype involves the human genome. Nonrandom chromosomal aberrations that are frequently observed in tumors are presented. Altered metabolic demands of the tumor cell are reflected in altered gene expressions of a wide range of enzymes and other proteins, and these changed enzyme patterns are described. The study of oncogenes increasingly suggests that they may be significant in certain cancers, and the assignment of these genes has been tabulated. The biochemical and metabolic changes observed in tumors are complex; studying the patterns and interactions of these changes will aid our genetic understanding of the origins and development of tumors.


title: Genetic variation in parameters reflecting immune competence of swine

text: Genetic variation in total and differential white blood cell (WBC) counts, phagocytic capacity of polymorphonuclear leukocytes (PMNL), virus induced interferon-α (IFN-α) production, mitogen induced proliferation and interleukin 2 (IL-2) production of mononuclear cells (MNC) in vitro was studied in blood collected from 124 Yorkshire piglets, aged 8 weeks. The piglets were the offspring from 12 sires and 31 dams. Data from an earlier experiment, including 96 piglets of seven sires and 24 dams, were added when estimating heritabilities for Con A induced proliferation and IL-2 production. The highest heritability (h(2)=0.87±0.41) was estimated for the total number of PMNL. Medium high heritabilities (h(2)=0.3−0.4) were estimated for the phagocytic capacity of PMNL, Con A induced proliferation and IL-2 production and the total number of WBC, while the heritability estimates were lower (h(2)=0.00−0.08±0.12) for the total number of lymphocytes, serum concentrations of Ig and IFN-α production. Pronounced differences between litters from various dams were found for total number of lymphocytes, IFN-α production, Con A induced proliferation and IL-2 production. The Con A induced proliferation was positively correlated (r=0.48, P<0.001) with the IL-2 production and both these parameters were correlated (r=0.44 and 0.37, respectively, P<0.001) to the virus induced IFN-α production. Despite these positive correlations, no parental offspring group was uniformly superior across all traits measured. However, the heritabilities estimated for the immune parameters are sufficiently high to be used as genetic markers in selection for general immune competence of swine.


title: Host Impairments in Patients with Neoplastic Diseases

text: Healthy individuals possess an immune system comprising physical barriers, innate and acquired immunity as well as the indigenous microflora that populate the body surfaces. The immune system maintains constant vigilance over the body at the cellular level as well as at the interface between the host integument and the resident microflora. However, neoplastic diseases and their treatment often lead to impaired immunity resulting in an increased risk of infections due to viruses, bacteria, fungi, and protozoa. This chapter explores the various aspects of host impairment focusing on the components of immunity and the interplay between them to explain why it is that these patients succumb to infections per se. In so doing, we hope that the reader will be better equipped to understand the risks patients face so as to anticipate potential infectious complications and implement appropriate measures to help attain successful remission of the neoplastic diseases and maintain the best quality of life for the patient.


title: HPLC Analysis and Purification of Peptides

text: High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic α-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins.


title: Yeast Two-Hybrid Screens: Improvement of Array-Based Screening Results by N- and C-terminally Tagged Fusion Proteins

text: Matrix-based yeast two-hybrid screens are an alternative to library-based screens. Recent improvements of matrix screens (also called array screens), use various pooling strategies as well as novel vectors that increase its efficiency while decreasing the false-negative rate, thus increasing reliability. In this chapter, we describe a screening strategy that systematically combines N- and C-terminal fusion proteins using a recently developed vector system.


title: New Strategies in Drug Discovery

text: Gene identification followed by determination of the expression of genes in a given disease and understanding of the function of the gene products is central to the drug discovery process. The ability to associate a specific gene with a disease can be attributed primarily to the extraordinary progress that has been made in the areas of gene sequencing and information technologies. Selection and validation of novel molecular targets have become of great importance in light of the abundance of new potential therapeutic drug targets that have emerged from human gene sequencing. In response to this revolution within the pharmaceutical industry, the development of high-throughput methods in both biology and chemistry has been necessitated. Further, the successful translation of basic scientific discoveries into clinical experimental medicine and novel therapeutics is an increasing challenge. As such, a new paradigm for drug discovery has emerged. This process involves the integration of clinical, genetic, genomic, and molecular phenotype data partnered with cheminformatics. Central to this process, the data generated are managed, collated, and interpreted with the use of informatics. This review addresses the use of new technologies that have arisen to deal with this new paradigm.


title: Tumor necrosis factor induces expression of MHC class I antigens on mouse astrocytes

text: The effect of tumor necrosis factor (TNF) on expression of major histocompatibility complex (MHC) antigens was examined in mouse glial cells in vitro. TNF induced MHC class I, but not class II, antigen expression on the surface of astrocytes but not on oligodendrocytes. Glial cells do not normally express detectable amounts of MHC antigens. Thus TNF may play a role in the immunopathogenesis of neurologic diseases that involve MHC class I-restricted reactions.


title: Evolutionary Influences in Arboviral Disease

text: Arthropod-borne viruses (arboviruses) generally require horizontal transmission by arthropod vectors among vertebrate hosts for their natural maintenance. This requirement for alternate replication in disparate hosts places unusual evolutionary constraints on these viruses, which have probably limited the evolution of arboviruses to only a few families of RNA viruses (Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, and Orthomyxoviridae) and a single DNA virus. Phylogenetic studies have suggested the dominance of purifying selection in the evolution of arboviruses, consistent with constraints imposed by differing replication environments and requirements in arthropod and vertebrate hosts. Molecular genetic studies of alphaviruses and flaviviruses have also identified several mutations that effect differentially the replication in vertebrate and mosquito cells, consistent with the view that arboviruses must adopt compromise fitness characteristics for each host. More recently, evidence of positive selection has also been obtained from these studies. However, experimental model systems employing arthropod and vertebrate cell cultures have yielded conflicting conclusions on the effect of alternating host infections, with host specialization inconsistently resulting in fitness gains or losses in the bypassed host cells. Further studies using in vivo systems to study experimental arbovirus evolution are critical to understanding and predicting disease emergence, which often results from virus adaptation to new vectors or amplification hosts. Reverse genetic technologies that are now available for most arbovirus groups should be exploited to test assumptions and hypotheses derived from retrospective phylogenetic approaches.


title: Animal Challenge Models of Henipavirus Infection and Pathogenesis

text: The henipaviruses, Hendra virus (HeV), and Nipah virus (NiV), are enigmatic emerging pathogens that causes severe and often fatal neurologic and/or respiratory disease in both animals and humans. Amongst people, case fatality rates range between 40 and 75% and there are no vaccines or treatments approved for human use. A number of species of animals including guinea pigs, hamsters, cats, ferrets, pigs, and African green monkeys have been employed as animal models of human henipavirus infection. Here, we review the development of animal models for henipavirus infection, discuss the pathology and pathogenesis of these models, and assess the utility of each model to recapitulate important aspects of henipavirus-mediated disease seen in humans.


title: Chemical and Genetic Tools to Explore S1P Biology

text: The zwitterionic lysophospholipid Sphingosine 1-Phosphate (S1P) is a pleiotropic mediator of physiology and pathology. The synthesis, transport, and degradation of S1P are tightly regulated to ensure that S1P is present in the proper concentrations in the proper location. The binding of S1P to five G protein-coupled S1P receptors regulates many physiological systems, particularly the immune and vascular systems. Our understanding of the functions of S1P has been aided by the tractability of the system to both chemical and genetic manipulation. Chemical modulators have been generated to affect most of the known components of S1P biology, including agonists of S1P receptors and inhibitors of enzymes regulating S1P production and degradation. Genetic knockouts and manipulations have been similarly engineered to disrupt the functions of individual S1P receptors or enzymes involved in S1P metabolism. This chapter will focus on the development and utilization of these chemical and genetic tools to explore the complex biology surrounding S1P and its receptors, with particular attention paid to the in vivo findings that these tools have allowed for.


title: Prioritization of Pandemic Influenza Vaccine: Rationale and Strategy for Decision Making

text: Few catastrophes can compare with the global impact of a severe influenza pandemic. The 1918–1919 pandemic was associated with more than 500,000 deaths in the USA and an estimated 20–40 million deaths worldwide, though some place the global total much higher. In an era when infectious disease mortality had been steadily decreasing, the 1918–1919 pandemic caused a large spike in overall population mortality, temporarily reversing decades of progress. The US Department of Health and Human Services, extrapolating from the 1918–1919 pandemic to the current US population size and demographics, has estimated that a comparable pandemic today would result in almost two million deaths. Vaccination is an important component of a pandemic response. Public health measures such as reduction of close contacts with others, improved hygiene, and respiratory protection with facemasks or respirators can reduce the risk of exposure and illness (Germann et al. 2006; Ferguson et al. 2006), but would not reduce susceptibility among the population. Prophylaxis with antiviral medications also may prevent illness but depends on the availability of large antiviral drug stockpiles and also does not provide long-term immunity. By contrast, immunization with a well-matched pandemic vaccine would provide active immunity and represent the most durable pandemic response. However, given current timelines for the development of a pandemic influenza vaccine and its production capacity, vaccine is likely not to be available in sufficient quantities to protect the entire population before pandemic outbreaks occur, and thus potentially limited stocks may need to be prioritized. This chapter reviews information on influenza vaccine production capacity, describes approaches used in the USA to set priorities for vaccination in the setting of limited supply, and presents a proposed strategy for prioritization.


title: Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future

text: Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo.


title: Ricin and Shiga Toxins: Effects on Host Cell Signal Transduction

text: Shiga toxins and ricin are potent inhibitors of protein synthesis. In addition to causing inhibition of protein synthesis, these toxins activate proinflammatory signaling cascades that may contribute to the severe diseases associated with toxin exposure. Treatment of cells with Shiga toxins and ricin have been shown to activate a number of signaling pathways including those associated with the ribotoxic stress response, Nuclear factor kappa B activation, inflammasome activation, the unfolded protein response, mTOR signaling, hemostasis, and retrograde trafficking. In this chapter, we review our current understanding of these signaling pathways as they pertain to intoxication by Shiga toxins and ricin.


title: Roles of Drug Transporters in Blood-Retinal Barrier

text: Blood-retinal barrier (BRB) includes inner BRB (iBRB) and outer BRB (oBRB), which are formed by retinal capillary endothelial (RCEC) cells and by retinal pigment epithelial (RPE) cells in collaboration with Bruch’s membrane and the choriocapillaris, respectively. Functions of the BRB are to regulate fluids and molecular movement between the ocular vascular beds and retinal tissues and to prevent leakage of macromolecules and other potentially harmful agents into the retina, keeping the microenvironment of the retina and retinal neurons. These functions are mainly attributed to absent fenestrations of RCECs, tight junctions, expression of a great diversity of transporters, and coverage of pericytes and glial cells. BRB existence also becomes a reason that systemic administration for some drugs is not suitable for the treatment of retinal diseases. Some diseases (such as diabetes and ischemia-reperfusion) impair BRB function via altering tight junctions, RCEC death, and transporter expression. This chapter will illustrate function of BRB, expressions and functions of these transporters, and their clinical significances.


title: Prioritization of High Consequence Viruses to Improve European Laboratory Preparedness for Cross-Border Health Threats

text: Highly infectious diseases can spread rapidly across borders through travel or trade, and international coordination is essential to a prompt and efficient response by public health laboratories. Therefore, developing strategies to identify priorities for a rational allocation of resources for research and surveillance has been the focus of a large body of research in recent years. This paper describes the activities and the strategy used by a European-wide consortium funded by the European Commission, named EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level), for the selection of high-threat pathogens with cross-border potential that will become the focus of its preparedness activities. The approach used is based on an objective scoring system, a close collaboration with other networks dealing with highly infection diseases, and a diagnostic gaps analysis. The result is a tool that is simple, objective and adaptable, which will be used periodically to re-evaluate activities and priorities, representing a step forward towards a better response to infectious disease emergencies.


title: Mapping the Protein–Protein Interactome Networks Using Yeast Two-Hybrid Screens

text: The yeast two-hybrid system (Y2H) is a powerful method to identify binary protein–protein interactions in vivo. Here we describe Y2H screening strategies that use defined libraries of open reading frames (ORFs) and cDNA libraries. The array-based Y2H system is well suited for interactome studies of small genomes with an existing ORFeome clones preferentially in a recombination based cloning system. For large genomes, pooled library screening followed by Y2H pairwise retests may be more efficient in terms of time and resources, but multiple sampling is necessary to ensure comprehensive screening. While the Y2H false positives can be efficiently reduced by using built-in controls, retesting, and evaluation of background activation; implementing the multiple variants of the Y2H vector systems is essential to reduce the false negatives and ensure comprehensive coverage of an interactome.


title: Advances in Vaccines

text: [Image: see text]


title: Exploring the Ligand-Protein Networks in Traditional Chinese Medicine: Current Databases, Methods and Applications

text: While the concept of “single component–single target” in drug discovery seems to have come to an end, “Multi-component–multi-target” is considered to be another promising way out in this field. The Traditional Chinese Medicine (TCM), which has thousands of years’ clinical application among China and other Asian countries, is the pioneer of the “Multi-component–multi-target” and network pharmacology. Hundreds of different components in a TCM prescription can cure the diseases or relieve the patients by modulating the network of potential therapeutic targets. Although there is no doubt of the efficacy, it is difficult to elucidate convincing underlying mechanism of TCM due to its complex composition and unclear pharmacology. Without thorough investigation of its potential targets and side effects, TCM is not able to generate large-scale medicinal benefits, especially in the days when scientific reductionism and quantification are dominant. The use of ligand-protein networks has been gaining significant value in the history of drug discovery while its application in TCM is still in its early stage. This article firstly surveys TCM databases for virtual screening that have been greatly expanded in size and data diversity in recent years. On that basis, different screening methods and strategies for identifying active ingredients and targets of TCM are outlined based on the amount of network information available, both on sides of ligand bioactivity and the protein structures. Furthermore, applications of successful in silico target identification attempts are discussed in details along with experiments in exploring the ligand-protein networks of TCM. Finally, it will be concluded that the prospective application of ligand-protein networks can be used not only to predict protein targets of a small molecule, but also to explore the mode of action of TCM.


title: Beyond Double-Stranded RNA-Type I IFN Induction by 3pRNA and Other Viral Nucleic Acids

text: Production of type I IFN is the key response to viral infection. Since the discovery of type I IFNs in 1957, long double-stranded RNA formed during replication of many viruses was thought to be responsible for type I IFN induction, and for decades double-stranded RNA-activated protein kinase (PKR) was thought to be the receptor. Recently, this picture has dramatically changed. It now became evident that not PKR but two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, and two cytosolic helicases, RIG-I and MDA-5, are responsible for the majority of type I IFNs induced upon recognition of viral nucleic acids. In this review, we focus on the molecular mechanisms by which those innate immune receptors detect viral infection. Based on the recent progress in the field, we now know that TLR7, TLR9, and RIG-I do not require long double-stranded RNA for type I IFN induction.


title: Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System

text: A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [(35)S]GTPγS assays). The human H(1)R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH(1)R and insect cell Gα(q). By contrast, functional expression of the hH(2)R required the generation of an hH(2)R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH(2)R to the long (Gsα(L)) or short (Gsα(S)) splice variant of Gα(s) resulted in comparable constitutive hH(2)R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH(1)R/hH(2)R and their guinea pig orthologues gpH(1)R/gpH(2)R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH(3)R with Gα(i1), Gα(i2), Gα(i3), and Gα(i/o) in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li(+), Na(+), K(+)) and anions (Cl(−), Br(−), I(−)) revealed that anions with large radii most efficiently stabilize the inactive hH(3)R state. Potential sodium binding sites in the hH(3)R protein were analyzed by expressing specific hH(3)R mutants in Sf9 cells. In contrast to the hH(3)R, the hH(4)R preferentially couples to co-expressed Gα(i2) in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH(4)R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH(4)R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH(4)R. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of H(x)R pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.


title: Mucosal Immunization and Adjuvants

text: The goal of the influenza vaccine is to prevent influenza virus infection and control the yearly seasonal epidemic and pandemic. However, the presently available parenteral influenza vaccine induces only systemic humoral immunity, which does not prevent influenza virus infection on the mucosal surface. Secretary IgA antibodies play an important role in preventing natural infection. Moreover, the IgA antibody response mediates cross-protection against variant viruses in animal models. Thus, a mucosal influenza vaccine that induces mucosal immunity would be a powerful tool to protect individuals from the influenza virus. Although the function of the mucosal immune system, especially in the respiratory tract, is not completely understood, there are several studies underway to develop mucosal influenza vaccines. Here, we will review current knowledge concerning the induction of IgA, the role of B-cell production of influenza virus specific IgA antibodies in anti-influenza immunity, and the role of humoral memory responses induced upon vaccination.


title: In Silico Models for Ecotoxicity of Pharmaceuticals

text: Pharmaceuticals and their active metabolites are one of the significantly emerging environmental toxicants. The major routes of entry of pharmaceuticals into the environment are industries, hospitals, or direct disposal of unwanted or expired drugs made by the patient. The most important and distinct features of pharmaceuticals are that they are deliberately designed to have an explicit mode of action and designed to exert an effect on humans and other living systems. This distinctive feature makes pharmaceuticals and their metabolites different from other chemicals, and this necessitates the evaluation of the direct effects of pharmaceuticals in various environmental compartments as well as to living systems. In this background, the alarming situation of ecotoxicity of diverse pharmaceuticals have forced government and nongovernment regulatory authorities to recommend the application of in silico methods to provide quick information about the risk assessment and fate properties of pharmaceuticals as well as their ecological and indirect human health effects. This chapter aims to offer information regarding occurrence of pharmaceuticals in the environment, their persistence, environmental fate, and toxicity as well as application of in silico methods to provide information about the basic risk management and fate prediction of pharmaceuticals in the environment. Brief ideas about toxicity endpoints, available ecotoxicity databases, and expert systems employed for rapid toxicity predictions of ecotoxicity of pharmaceuticals are also discussed.


title: Predicting Synthetic Gene Networks

text: Synthetic biology aims at designing and building new biological functions in living organisms. The complexity of cellular regulation (regulatory, metabolic, and signaling interactions, and their coordinated action) can be tackled via the development of quantitative mathematical models. These models are useful to test biological hypotheses and observations, and to predict the possible behaviors of a synthetic network. Indeed, synthetic biology uses such models to design synthetic networks, prior to their construction in the cell, to perform specific tasks, or to change a biological process in a desired way. The synthetic network is built by assembling biological “parts” taken from different systems; therefore it is fundamental to identify, isolate, and test regulatory motifs which occur frequently in biological pathways. In this chapter, we describe how to model and predict the behavior of synthetic networks in two difference cases: (1) a synthetic network composed of five genes regulating each other through a variety of regulatory interactions in the yeast Saccharomyces cerevisiae (2) a synthetic transcriptional positive feedback loop stably integrated in Human Embryonic Kidney 293 cells (HEK293).


title: Therapeutic Applications

text: The current treatments offered to patients with chronic respiratory diseases are being re-evaluated based on the loss of potency during long-term treatments or because they only provide significant clinical benefits to a subset of the patient population. For instance, glucocorticoids are considered the most effective anti-inflammatory therapies for chronic inflammatory and immune diseases, such as asthma. But they are relatively ineffective in asthmatic smokers, and patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). As such, the pharmaceutical industry is exploring new therapeutic approaches to address all major respiratory diseases. The previous chapters demonstrated the widespread influence of purinergic signaling on all pulmonary functions and defense mechanisms. In Chap. 8, we described animal studies which highlighted the critical role of aberrant purinergic activities in the development and maintenance of chronic airway diseases. This last chapter covers all clinical and pharmaceutical applications currently developed based on purinergic receptor agonists and antagonists. We use the information acquired in the previous chapters on purinergic signaling and lung functions to scrutinize the preclinical and clinical data, and to realign the efforts of the pharmaceutical industry.


title: Sensory Nerves and Airway Irritability

text: The lung, like many other organs, is innervated by a variety of sensory nerves and by nerves of the parasympathetic and sympathetic nervous systems that regulate the function of cells within the respiratory tract. Activation of sensory nerves by both mechanical and chemical stimuli elicits a number of defensive reflexes, including cough, altered breathing pattern, and altered autonomic drive, which are important for normal lung homeostasis. However, diseases that afflict the lung are associated with altered reflexes, resulting in a variety of symptoms, including increased cough, dyspnea, airways obstruction, and bronchial hyperresponsiveness. This review summarizes the current knowledge concerning the physiological role of different sensory nerve subtypes that innervate the lung, the factors which lead to their activation, and pharmacological approaches that have been used to interrogate the function of these nerves. This information may potentially facilitate the identification of novel drug targets for the treatment of respiratory disorders such as cough, asthma, and chronic obstructive pulmonary disease.


title: Pulmonary Fibrosis

text: Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing lung disease limited to the lungs and associated with the histologic appearance of usual interstitial pneumonia (UIP) on surgical lung biopsy. The estimated prevalence in the United States is between 35,000 and 55,000 cases, and evidence suggests that the prevalence is increasing for IPF. Risk factors associated with pulmonary fibrosis include smoking, environmental exposures, gastroesophageal reflux disease, commonly prescribed drugs, diabetes mellitus, infectious agents, and genetic factors. The diagnosis requires a careful history and physical examination, characteristic physiological and radiological studies, and, in some cases, a surgical lung biopsy. The natural history of IPF is not known, but evidence supports the concept of a continuum of idiopathic interstitial pneumonias that may overlap in time. Most patients with IPF succumb to respiratory failure, cardiovascular disease, lung cancer, pulmonary embolism, infection, and other health problems. The median survival time for patients with IPF is less than 3 yr. Factors that predict poor outcome include older age, male gender, severe dyspnea, history of cigarette smoking, severe loss of lung function, appearance and severity of fibrosis on radiological studies, lack of response to therapy, and prominent fibroblastic foci on histopathologic evaluation. Conventional therapy (corticosteroids, azathioprine, cyclophosphamide) provides only marginal benefit. Lung transplantation should be considered for patients with IPF refractory to medical therapy. In light of the poor prognosis and lack of response to available anti-inflammatory therapy, alternative approaches to therapy are being pursued. Emerging strategies to treat patients with IPF include agents that inhibit epithelial injury or enhance repair, anticytokine approaches, agents that inhibit fibroblast proliferation or induce fibroblast apoptosis, and other novel approaches.


title: Human Monoclonal Antibodies from Transgenic Mice

text: Since the 1986 regulatory approval of muromonomab-CD3, a mouse monoclonal antibody (MAb) directed against the T cell CD3ε antigen, MAbs have become an increasingly important class of therapeutic compounds in a variety of disease areas ranging from cancer and autoimmune indications to infectious and cardiac diseases. However, the pathway to the present acceptance of therapeutic MAbs within the pharmaceutical industry has not been smooth. A major hurdle for antibody therapeutics has been the inherent immunogenicity of the most readily available MAbs, those derived from rodents. A variety of technologies have been successfully employed to engineer MAbs with reduced immunogenicity. Implementation of these antibody engineering technologies involves in vitro optimization of lead molecules to generate a clinical candidate. An alternative technology, involving the engineering of strains of mice to produce human instead of mouse antibodies, has been emerging and evolving for the past two decades. Now, with the 2006 US regulatory approval of panitumumab, a fully human antibody directed against the epidermal growth factor receptor, transgenic mice expressing human antibody repertoires join chimerization, CDR grafting, and phage display technologies, as a commercially validated antibody drug discovery platform. With dozens of additional transgenic mouse-derived human MAbs now in clinical development, this new drug discovery platform appears to be firmly established within the pharmaceutical industry.


title: Biosafety and Biohazards: Understanding Biosafety Levels and Meeting Safety Requirements of a Biobank

text: When it comes to biobanking and working with different types of laboratory specimens, it is important to understand potential biohazards to ensure safety of the operator and laboratory personnel. Biological safety levels (BSL) are a series of designations used to inform laboratory personnel about the level of biohazardous risks in a laboratory setting. There are a total of four levels ranked in order of increasing risk as stipulated by the Center of Disease Control and Prevention (CDC) (Biosafety in microbiological and biomedical laboratories, 5th edn. HHS publication no. (CDC) 21-1112. https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf. Accessed 2 Jan 2016, 2009). We will address the main distinctions between these levels including briefly introducing hazards characteristics that classify biohazardous agents, as well as define the essentials in meeting safety requirements.


Question Sets and Answers


title: Impact of Pharmaceuticals on the Environment: Risk Assessment Using QSAR Modeling Approach

text: An extensive use of pharmaceuticals and the widespread practices of their erroneous disposal measures have made these products contaminants of emerging concern (CEC). Especially, active pharmaceutical ingredients (APIs) are ubiquitously detected in surface water and soil, mainly in the aquatic compartment, where they do affect the living systems. Unfortunately, there is a huge gap in the availability of ecotoxicological data on pharmaceuticals’ environmental behavior and ecotoxicity which force EMEA (European Medicines Agency) to release guidelines for their risk assessment. In silico modeling approaches are vital tools to exploit the existing information to rapidly emphasize the potentially most hazardous and toxic pharmaceuticals and prioritize the most environmentally hazardous ones for focusing further on their experimental studies. The quantitative structure–activity relationship (QSAR) models are capable of predicting missing properties for toxic end-points required to prioritize existing, or newly synthesized chemicals for their potential hazard. This chapter reviews the information regarding occurrence and impact of pharmaceuticals and their metabolites in the environment along with their persistence, environmental fate, risk assessment, and risk management. A bird’s eye view about the necessity of in silico methods for fate prediction of pharmaceuticals in the environment as well as existing successful models regarding ecotoxicity of pharmaceuticals are discussed. Available toxicity endpoints, ecotoxicity databases, and expert systems frequently used for ecotoxicity predictions of pharmaceuticals are also reported. The overall discussion justifies the requirement to build up additional in silico models for quick prediction of ecotoxicity of pharmaceuticals economically, without or involving only limited animal testing.


title: Mass Vaccination and Surveillance/Containment in the Eradication of Smallpox

text: The Smallpox Eradication Program, initiated by the WHO in 1966, was originally based on mass vaccination. The program emphasized surveillance from the beginning, largely to track the success of the program and further our understanding of the epidemiology of the disease. Early observations in West Africa, bolstered by later data from Indonesia and the Asian subcontinent, showed that smallpox did not spread rapidly, and outbreaks could be quickly controlled by isolation of patients and vaccination of their contacts. Contacts were usually easy to find because transmission of smallpox usually required prolonged face-to-face contact. The emphasis therefore shifted to active searches to find cases, coupled with contact tracing, rigorous isolation of patients, and vaccination and surveillance of contacts to contain outbreaks. This shift away from mass vaccination resulted in an acceleration of the program’s success.


title: Intranasal Delivery of Antiviral siRNA

text: Intranasal administration of synthetic siRNA is an effective modality of RNAi delivery for the prevention and therapy of respiratory diseases, including pulmonary infections. Vehicles used for nasal siRNA delivery include established as well as novel reagents, many of which have been recently optimized. In general, they all promote significant uptake of siRNA into the lower respiratory tract, including the lung. When properly designed and optimized, these siRNAs offer significant protection against respiratory viruses such as influenza virus, parainfluenza virus and respiratory syncytial virus (RSV). Nasally administered siRNA remains within the lung and does not access systemic blood flow, as judged by its absence in other major organs such as liver, heart, kidney, and skeletal muscle. Adverse immune reaction is generally not encountered, especially when immunogenic and/or off-target siRNA sequences and toxic vehicles are avoided. In fact, siRNA against RSV has entered Phase II clinical trials in human with promising results. Here, we provide a standardized procedure for using the nose as a specific route for siRNA delivery into the lung of laboratory animals. It should be clear that this simple and efficient system has enormous potential for therapeutics.


title: Epidemiology of Infections in Cancer Patients

text: Although major advances in the care of cancer patients over the past several decades have resulted in improved survival, infectious complications remain a significant cause of morbidity and mortality. To successfully identify, treat, and prevent infections, a comprehensive understanding of risk factors that predispose to infection and of commonly encountered pathogens is necessary. In addition, clinicians must keep abreast of the changing epidemiology of infections in this population. As therapeutic modalities continue to evolve, as established pathogens become increasingly drug resistant, and as new pathogens are discovered, successful management of infections will continue to present challenges in the years to come.


title: Towards Keeping the Hippocratic Oath (Six Sigma)

text: INTRODUCTION AND OBJECTIVE: Stone disease has a fascinating history and a bright future. Based upon our current understanding of this rather complex disease, the knowledge that is currently being generated in the basic science of stone disease offers the promise of minimizing patient suffering. But the technology and information are expanding at incomprehensible rates. METHODS: This is a history of technology and information systems themselves. This history is absolutely fascinating and deserves a spot in the lexicon of the history of urolithiasis because it is the future of management of this ancient malady and best hope for the future. A review of the errors of complex systems and the application to medicine and surgery centers on the notion of six sigma (six standard deviations from the norm). This is a statistical reference to improving outcomes. RESULTS: This chapter jumps to the history of modern electronics, computers, the rise of the Internet, and artificial intelligence and how it might be applied to urolithiasis. By necessity, complex systems in turn tend to focus on the complications and unintended consequences of the technology. Therefore, some attention will be paid to complications, medical malpractice, and the notion of human error. CONCLUSIONS: This concluding chapter brings us up-to-date in the technologies that will evolve and modify our current perceptions of stone disease. This includes the surgical and medical methods that will evolve in conjunction with the technologies that will drive all of the future of medicine, intelligent technology. The notion of convergence will be developed and presented and why such luminaries as Bill Joy have become so pessimistic about the future.


title: Identification and Analysis of Frameshift Sites

text: There are several ways that genes may encode alternative products. The most widely recognized mechanism is alternative splicing. However, genes may also employ noncanonical translational events to produce such products. Some of these mechanisms operate at the level of translational initiation. In prokaryotes, genes may include alternative ribosome-binding sites directing the synthesis of products that differ at the N terminus. In eukaryotes, in which ribosome-binding sites do not exist, leaky scanning allows the same kind of variation. Noncanonical elongation events can also generate products that differ at their C terminus (1–3). Such events include programmed readthrough of translational termination codons (4,5) translational frameshifts (6–9), and translational hops (10,11). In each case, the ribosome fails to follow normal rules of decoding, leading to the synthesis of a protein that is not encoded, in the normal sense, in the DNA. In this chapter, we will describe the methods employed in the identification and analysis of programmed translational frameshift sites, including their discovery, measurement of the efficiency of the events, and determination of the mechanism of the frameshift.


title: Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays

text: Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.


title: Role of Podocyte Injury in Glomerulosclerosis

text: Finding new therapeutic targets of glomerulosclerosis treatment is an ongoing quest. Due to a living environment of various stresses and pathological stimuli, podocytes are prone to injuries; moreover, as a cell without proliferative potential, loss of podocytes is vital in the pathogenesis of glomerulosclerosis. Thus, sufficient understanding of factors and underlying mechanisms of podocyte injury facilitates the advancement of treating and prevention of glomerulosclerosis. The clinical symptom of podocyte injury is proteinuria, sometimes with loss of kidney functions progressing to glomerulosclerosis. Injury-induced changes in podocyte physiology and function are actually not a simple passive process, but a complex interaction of proteins that comprise the anatomical structure of podocytes at molecular levels. This chapter lists several aspects of podocyte injuries along with potential mechanisms, including glucose and lipid metabolism disorder, hypertension, RAS activation, micro-inflammation, immune disorder, and other factors. These aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries.


title: Taking Bioinformatics to Systems Medicine

text: Systems medicine promotes a range of approaches and strategies to study human health and disease at a systems level with the aim of improving the overall well-being of (healthy) individuals, and preventing, diagnosing, or curing disease. In this chapter we discuss how bioinformatics critically contributes to systems medicine. First, we explain the role of bioinformatics in the management and analysis of data. In particular we show the importance of publicly available biological and clinical repositories to support systems medicine studies. Second, we discuss how the integration and analysis of multiple types of omics data through integrative bioinformatics may facilitate the determination of more predictive and robust disease signatures, lead to a better understanding of (patho)physiological molecular mechanisms, and facilitate personalized medicine. Third, we focus on network analysis and discuss how gene networks can be constructed from omics data and how these networks can be decomposed into smaller modules. We discuss how the resulting modules can be used to generate experimentally testable hypotheses, provide insight into disease mechanisms, and lead to predictive models. Throughout, we provide several examples demonstrating how bioinformatics contributes to systems medicine and discuss future challenges in bioinformatics that need to be addressed to enable the advancement of systems medicine.


title: ROS Signaling in the Pathogenesis of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS)

text: The generation of reactive oxygen species (ROS) plays an important role for the maintenance of cellular processes and functions in the body. However, the excessive generation of oxygen radicals under pathological conditions such as acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) leads to increased endothelial permeability. Within this hallmark of ALI and ARDS, vascular microvessels lose their junctional integrity and show increased myosin contractions that promote the migration of polymorphonuclear leukocytes (PMNs) and the transition of solutes and fluids in the alveolar lumen. These processes all have a redox component, and this chapter focuses on the role played by ROS during the development of ALI/ARDS. We discuss the origins of ROS within the cell, cellular defense mechanisms against oxidative damage, the role of ROS in the development of endothelial permeability, and potential therapies targeted at oxidative stress.


title: Challenges and Opportunities for Respiratory Syncytial Virus Vaccines

text: Respiratory syncytial virus (RSV) causes a significant proportion of the global burden of respiratory disease. Here we summarize the conclusions of a series of chapters written by investigators describing and interpreting what is known about the virology, clinical manifestations, immunity, pathogenesis, and epidemiology of RSV relevant to vaccine development. Several technological and conceptual advances have recently occurred that make RSV vaccine development more feasible, and this collected knowledge is intended to help inform and organize the future contributions of funding agencies, scientists, regulatory agencies, and policy makers that will be needed to achieve the goal of a safe, effective, and accessible vaccine to prevent RSV-associated disease.


title: Chromatin as a Target for the DNA-Binding Anticancer Drugs

text: Chemotherapy has been a major approach to treat cancer. Both constituents of chromatin, chromosomal DNA and the associated chromosomal histone proteins are the molecular targets of the anticancer drugs. Small DNA binding ligands, which inhibit enzymatic processes with DNA substrate, are well known in cancer chemotherapy. These drugs inhibit the polymerase and topoisomerase activity. With the advent in the knowledge of chromatin chemistry and biology, attempts have shifted from studies of the structural basis of the association of these drugs or small ligands (with the potential of drugs) with DNA to their association with chromatin and nucleosome. These drugs often inhibit the expression of specific genes leading to a series of biochemical events. An overview will be given about the latest understanding of the molecular basis of their action. We shall restrict to those drugs, synthetic or natural, whose prime cellular targets are so far known to be chromosomal DNA


title: The Preparation of Chicken Tracheal Organ Cultures for Virus Isolation, Propagation, and Titration

text: Chicken tracheal organ cultures (TOCs), comprising transverse sections of chick embryo trachea with beating cilia, have proved useful in the isolation of several respiratory viruses and as a viral assay system, using ciliostasis as the criterion for infection. A simple technique for the preparation of chicken tracheal organ cultures in glass test tubes, in which virus growth and ciliostasis can be readily observed, is described.


Emerging infectious diseases: the public’s view of the problem and what should be expected from the public health community


title: The Role of the Plant Nucleolus in Pre-mRNA Processing

text: The nucleolus is a multifunctional compartment of the eukaryotic nucleus. Besides its well-recognised role in transcription and processing of ribosomal RNA and the assembly of ribosomal subunits, the nucleolus has functions in the processing and assembly of a variety of RNPs and is involved in cell cycle control and senescence and as a sensor of stress. Historically, nucleoli have been tenuously linked to the biogenesis and, in particular, export of mRNAs in yeast and mammalian cells. Recently, data from plants have extended the functions in which the plant nucleolus is involved to include transcriptional gene silencing as well as mRNA surveillance and nonsense-mediated decay, and mRNA export. The nucleolus in plants may therefore have important roles in the biogenesis and quality control of mRNAs.


title: Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines

text: The ferret is a standard laboratory animal that can be accommodated in most animal facilities. While not susceptible to measles, ferrets are a natural host of canine distemper virus (CDV), the closely related carnivore morbillivirus. CDV infection in ferrets reproduces all clinical signs associated with measles in humans, including the typical rash, fever, general immunosuppression, gastrointestinal and respiratory involvement, and neurological complications. Due to this similarity, experimental CDV infection of ferrets is frequently used to assess the efficacy of novel vaccines, and to characterize pathogenesis mechanisms. In addition, direct intracranial inoculation of measles isolates from subacute sclerosing panencephali-tis (SSPE) patients results in an SSPE-like disease in animals that survive the acute phase. Since the advent of reverse genetics systems that allow the targeted manipulation of viral genomes, the model has been used to evaluate the contribution of the accessory proteins C and V, and signalling lymphocyte activation molecule (SLAM)-binding to immunosuppression and overall pathogenesis. Similarly produced green fluorescent protein-expressing derivatives that maintain parental virulence have been instrumental in the direct visualization of systemic dissemination and neuroinvasion. As more immunological tools become available for this model, its contribution to our understanding of morbillivirus—host interactions is expected to increase.


title: Animal Models of Airway Diseases

text: Over the past 20 years, the growing awareness that purinergic signaling events literally shape the immune and inflammatory responses to infection and allergic reactions warranted the development of animal models to assess their importance in vivo in acute lung injury and chronic airway diseases. The pioneer work conducted with the adenosine deaminase (ADA)-deficient mouse provided irrefutable evidence that excess adenosine (ADO) accumulating in the lungs of asthmatic patients, constitutes a powerful mediator of disease severity. These original studies launched the development of murine strains for the two major ectonucleotidases responsible for the generation of airway ADO from ATP release: CD39 and CD73. The dramatic acute lung injury and chronic lung complications, manifested by these knockout mice in response to allergens and endotoxin, demonstrated the critical importance of regulating the availability of ATP and ADO for their receptors. Therapeutic targets are currently evaluated using knockout mice and agonists/antagonists for each ADO receptor (A(1)R, A(2A)R, A(2B)R, and A(3)R) and the predominant ATP receptors (P2Y(2)R and P2X(7)R). This chapter provides an in-depth description of each in vivo study, and a critical view of the therapeutic potentials for the treatment of airway diseases.


title: Transmission in the Guinea Pig Model

text: The ability of an influenza virus to transmit efficiently from human-to-human is a major factor in determining the epidemiological impact of that strain. The use of a relevant animal model to identify viral determinants of transmission, as well as host and environmental factors affecting transmission efficiency, is therefore critical for public health. The characterization of newly emerging influenza viruses in terms of their potential to transmit in a mammalian host is furthermore an important part of pandemic risk assessment. For these reasons, a guinea pig model of influenza virus transmission was developed in 2006. The guinea pig provides an important alternative to preexisting models for influenza. Most influenza viruses do not readily transmit among mice. Ferrets, while highly relevant, are expensive and can be difficult to obtain in high numbers. Moreover, it is generally accepted that efforts to accurately model human disease are strengthened by the use of multiple animal species. Herein, we provide an overview of influenza virus infectivity, growth, and transmission in the guinea pig and highlight knowledge gained on the topic of influenza virus transmission using the guinea pig model.


title: Peptide Lipidation – A Synthetic Strategy to Afford Peptide Based Therapeutics

text: Peptide and protein aberrant lipidation patterns are often involved in many diseases including cancer and neurological disorders. Peptide lipidation is also a promising strategy to improve pharmacokinetic and pharmacodynamic profiles of peptide-based drugs. Self-adjuvanting peptide-based vaccines commonly utilise the powerful TLR2 agonist Pam(n)Cys lipid to stimulate adjuvant activity. The chemical synthesis of lipidated peptides can be challenging hence efficient, flexible and straightforward synthetic routes to access homogeneous lipid-tagged peptides are in high demand. A new technique coined Cysteine Lipidation on a Peptide or Amino acid (CLipPA) uses a ‘thiol-ene’ reaction between a cysteine and a vinyl ester and offers great promise due to its simplicity, functional group compatibility and selectivity. Herein a brief review of various synthetic strategies to access lipidated peptides, focusing on synthetic methods to incorporate a Pam(n)Cys motif into peptides, is provided.


title: Classification of Human Leukocyte Antigen (HLA) Supertypes

text: Identification of new antigenic peptides, derived from infectious agents or cancer cells, which bind to human leukocyte antigen (HLA) class I and II molecules, is of importance for the development of new effective vaccines capable of activating the cellular arm of the immune response. However, the barrier to the development of peptide-based vaccines with maximum population coverage is that the restricting HLA genes are extremely polymorphic resulting in a vast diversity of peptide-binding HLA specificities and a low population coverage for any given peptide–HLA specificity. One way to reduce this complexity is to group thousands of different HLA molecules into several so-called HLA supertypes: a classification that refers to a group of HLA alleles with largely overlapping peptide binding specificities. In this chapter, we focus on the state-of-the-art classification of HLA supertypes including HLA-I supertypes and HLA-II supertypes and their application in development of peptide-based vaccines.


title: BMT Settings, Infection and Infection Control

text: Despite improvements over the past several decades, infection remains a significant risk to all haematological patients receiving therapy. Those requiring allogeneic transplant and especially those that have HLA disparity or T-cell-depleted grafts have an even higher risk of infective complications due to delayed recovery of T- and B-cell function. Early identification with prompt effective treatment is paramount to improve all patients’ survival. Patient safety through robust adherence to hand hygiene and maintenance of the environment with cleaning and disinfection are the backbone of an effective preventative program. Basic nursing care and a sound knowledge base of the risks, presentation, diagnosis and treatment will improve patient care.


title: In Vivo Infection Model of Severe Influenza A Virus

text: The lung is constantly exposed to both environmental and microbial challenge. As a “contained” organ, it also constitutes an excellent “self-contained” tissue to examine inflammatory responses and cellular infiltration into a diseased organ. Influenza A virus (IAV) causes both mild and severe inflammation that is strain specific following infection of the lung epithelium that spreads to other cells of the lung environment. Here, we describe a method of intranasal inoculation of the lung with IAV that can be used as a preclinical model of infection. Mice can be monitored for clinical signs of infection and tissue and lung fluid collected for further analysis to dissect the immunological consequences of IAV infection. Importantly, this method can be modified to introduce other pathogens, therapies and environmental stimuli to examine immune responses in the lung.


title: Bats and Emerging Infections: An Ecological and Virological Puzzle

text: More than 200 viruses have been detected in bats. Some unique bat characteristics can explain the roles played in the maintenance and transmission of viruses: long phylogenetic history can have originated coevolution processes, great number of species are adapted to live in different environments, big mobility, long lifespan and gregarious behaviour of many species. To analyse zoonoses long longitudinal studies are needed with a multidisciplinary approximation to obtain the following eco-epidemiological data: colony size, number of bats per species, population structure, behaviour of each species, degree of contact between bats, social structure, remaining time of bats in the colony, colony type, foraging area, turnover rate of individuals, shelter temperature, relationship with other colonies and co-infection processes. These data allows assessing the epidemiological risk and which preventive measures are necessary to take. The structure and functionality of ecosystems are changing worldwide at an unprecedented rate and can modify the interactions between humans and infected bats. There are more or less local factors that can affect the emergence and spread of diseases (environmental alterations, changes in land use, human population growth, changes in human socioeconomic behavior or social structure, people mobility increase, trade increase, forest fires, extreme weather events, wars, breakdown in public health infrastructure, etc.). Twenty-three percent of all bat species in the world are decreasing. How does the regression of bat species affect the dynamic of viruses? The dichotomy between health risk and bat preservation is compatible with a preventive task based on more information and training.


title: Strategies Towards Protease Inhibitors for Emerging Flaviviruses

text: Infections with flaviviruses are a continuing public health threat. In addition to vaccine development and vector control, the search for antiviral agents that alleviate symptoms in patients are of considerable interest. Among others, the flaviviral protease NS2B-NS3 is a promising drug target to inhibit viral replication. Flaviviral proteases share a high degree of structural similarity and substrate-recognition profile, which may facilitate a strategy towards development of pan-flaviviral protease inhibitors. However, the success of various drug discovery attempts during the last decade has been limited by the nature of the viral enzyme as well as a lack of robust structural templates. Small-molecular, structurally diverse protease inhibitors have been reported to reach affinities in the lower micromolar range. Peptide-based, substrate-derived compounds are often nanomolar inhibitors, however, with highly compromised drug-likeness. With some exceptions, the antiviral cellular activity of most of the reported compounds have been patchy and insufficient for further development. Recent progress has been made in the elucidation of inhibitor binding using different structural methods. This will hopefully lead to more rational attempts for the identification of various lead compounds that may be successful in cellular assays, animal models and ultimately translated to patients.


title: Sequencing DNA Amplified Directly from a Bacterial Colony

text: A few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (PCR) amplification procedure to identify and orient a plasmid insert (1,2). By combining this procedure with one in which asymmetrically amplified DNA is used for sequencing (ref. 3 and Fig. 3), we have demonstrated that DNA amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template DNA is used for amplification (ref.4 and Fig. 2). By end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. Inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded DNA sequence. The procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day.


title: Regulation and Function of NK and T Cells During Dengue Virus Infection and Vaccination

text: The focus of this review is to discuss findings in the last 10 years that have advanced our understanding of human NK cell responses to dengue virus. We will review recently identified interactions of activating and inhibitory receptors on NK cells with dengue virus, human NK responses to natural dengue infection and highlight possible interactions by which NK cells may shape adaptive immune responses. T cell responses to natural dengue infection will be reviewed by Laura Rivino in Chap. 10.1007/978-981-10-8727-1_17. With the advent of numerous dengue vaccine clinical trials, we will also review T and NK cell immune responses to dengue virus vaccination. As our understanding of the diverse functions of NK cell has advanced, it has become increasingly clear that human NK cell responses to viral infections are more complicated than initially recognized.


title: The Applicability of Mouse Models to the Study of Human Disease

text: The laboratory mouse Mus musculus has long been used as a model organism to test hypotheses and treatments related to understanding the mechanisms of disease in humans; however, for these experiments to be relevant, it is important to know the complex ways in which mice are similar to humans and, crucially, the ways in which they differ. In this chapter, an in-depth analysis of these similarities and differences is provided to allow researchers to use mouse models of human disease and primary cells derived from these animal models under the most appropriate and meaningful conditions. Although there are considerable differences between mice and humans, particularly regarding genetics, physiology, and immunology, a more thorough understanding of these differences and their effects on the function of the whole organism will provide deeper insights into relevant disease mechanisms and potential drug targets for further clinical investigation. Using specific examples of mouse models of human lung disease, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, this chapter explores the most salient features of mouse models of human disease and provides a full assessment of the advantages and limitations of these models, focusing on the relevance of disease induction and their ability to replicate critical features of human disease pathophysiology and response to treatment. The chapter concludes with a discussion on the future of using mice in medical research with regard to ethical and technological considerations.


title: Generating Inhibitors of P-Glycoprotein: Where to, Now?

text: The prominent role for the drug efflux pump ABCB1 (P-glycoprotein) in mediating resistance to chemotherapy was first suggested in 1976 and sparked an incredible drive to restore the efficacy of anticancer drugs. Achieving this goal seemed inevitable in 1982 when a series of calcium channel blockers were demonstrated to restore the efficacy of chemotherapy agents. A large number of other compounds have since been demonstrated to restore chemotherapeutic sensitivity in cancer cells or tissues. Where do we stand almost three decades since the first reports of ABCB1 inhibition? Unfortunately, in the aftermath of extensive fundamental and clinical research efforts the situation remains gloomy. Only a small handful of compounds have reached late stage clinical trials and none are in routine clinical usage to circumvent chemoresistance. Why has the translation process been so ineffective? One factor is the multifactorial nature of drug resistance inherent to cancer tissues; ABCB1 is not the sole factor. However, expression of ABCB1 remains a significant negative prognostic indicator and is closely associated with poor response to chemotherapy in many cancer types. The main difficulties with restoration of sensitivity to chemotherapy reside with poor properties of the ABCB1 inhibitors: (1) low selectivity to ABCB1, (2) poor potency to inhibit ABCB1, (3) inherent toxicity and/or (4) adverse pharmacokinetic interactions with anticancer drugs. Despite these difficulties, there is a clear requirement for effective inhibitors and to date the strategies for generating such compounds have involved serendipity or simple chemical syntheses. This chapter outlines more sophisticated approaches making use of bioinformatics, combinatorial chemistry and structure informed drug design. Generating a new arsenal of potent and selective ABCB1 inhibitors offers the promise of restoring the efficacy of a key weapon in cancer treatment – chemotherapy.


title: Design of Gene Constructs for Transgenic Maize

text: The first step of any maize transformation project is to select gene expression elements that will make up an effective construct. When designing a gene construct, one must have a full understanding of the different expression elements that are currently available and of the strategies that have been successfully used to overcome obstacles in past. In this chapter, we discuss several major classes of expression elements that have been used for maize transformation, including promoters, introns, and untranslated regions. We also discuss several strategies for further improving transgene expression levels, such as optimization of codon usage, removal of deleterious sequences, addition of signal sequences for subcellular protein targeting, and use of elements to reduce position effects. We hope that this chapter can serve as a general guideline to help researchers, especially beginners in the field, to design a gene construct that will have the maximum potential for gene expression.


title: Using ASMase Knockout Mice to Model Human Diseases

text: Acid sphingomyelinase (ASMase) is a key initiator of sphingomyelin/ceramide signal transduction activated by many stress stimuli. Over the past two decades, much progress has been made in defining the clinical relevance of sphingomyelin/ceramide signaling in numerous diseases using ASMase knockout mice. Organs that operate this pathway are numerous and the disease states regulated are diverse, with ceramide generation governing injury in tumor, gut, ovary, brain, lung, heart, liver, and during infection. This chapter emphasizes evolutionary conservation of sphingolipid stress signaling and mammalian adaptations that permit transduction of organotypic responses. Recognition that the sphingomyelin/ceramide transducer calibrates extent of tissue injury, ultimately acting as a molecular switch that determines organ fate, is driving development of new pharmacologic concepts and tools to intervene therapeutically.


title: Viral Forecasting, Pathogen Cataloging, and Disease Ecosystem Mapping: Measuring Returns on Investments

text: Infectious disease emergence into humans from animals or the environment occurs primarily due to genetic changes in the microbe through mutation or re-assortment making it either more transmissible or virulent or through a change in the disease “ecosystem”. Research into infectious disease emergence can be grouped into different strategic approaches. One strategic approach is to study a specific or model disease system to understand the ecology of an infectious disease and how is transmitted and propagated through the environment and different hosts and then extrapolate that disease system knowledge to related pathogens. The other strategic approach follows the genomics and phylogenetics—tracking how pathogens are evolving and changing at the amino acid level. Here we argue that for understanding complex zoonotic diseases and for the purposes of preventing emergence and re-emergence into humans, that the Return on Investment be considered for the best research strategy.


title: Molecular Basis of a Pandemic of Avian-Type Influenza Virus

text: Despite heroic efforts to prevent the emergence of an influenza pandemic, avian influenza A virus has prevailed by crossing the species barriers to infect humans worldwide, occasionally with morbidity and mortality at unprecedented levels, and the virus later usually continues circulation in humans as a seasonal influenza virus, resulting in health-social-economic problems each year. Here, we review current knowledge of influenza viruses, their life cycle, interspecies transmission, and past pandemics and discuss the molecular basis of pandemic acquisition, notably of hemagglutinin (lectin) acting as a key contributor to change in host specificity in viral infection.


title: Cellular Therapy Models: Ex Vivo Chimera Model by Cell Fusion

text: Cell fusion is a ubiquitous process fundamental to physiological and pathophysiological events common to multiple cell types and species. Performed ex vivo, cell fusion is a versatile research and therapeutic tool for gene mapping, antibody production, discovering new mechanisms in biological processes, inventing alternative therapies for cell reprogramming, restoring organ function, and creating cellular therapeutics for cancer treatment. Cell fusion can be successfully applied by creating cellular therapeutic of donor – recipient chimeric cell (DRCC) in the field of solid organ and vascularized composite allotransplantation (VCA). Immunomodulatory DRCC therapy has the potential to reduce or even eliminate the need for toxic, life-long immunosuppression and to prevent both acute and chronic rejection. This innovative VCA treatment is a combination of ex vivo created chimeric cell therapy with a short-term selective protocol of monoclonal antibody and Cyclosporine A. The utilization of short-term immunosuppressive protocol will provide the opportunity for chimeric cell engraftment, proliferation, and re-education of recipient’s immune system resulting in prolongation of allograft survival. The use of chimeric cells, as a supportive treatment for VCA, would improve the conditions of severely disfigured patients by offering safe alternative approach and providing better functional and aesthetic results compared to standard reconstructive procedures. This chapter summarizes the phenomenon, current discoveries, and advancements in the field of cell fusion, as well as introduces ex vivo creation of chimeric cells and presents potential benefits of chimeric cell-based protocols. Successful application of chimeric cell protocol in VCA experimental models will advance the field of reconstructive transplantation towards clinical trials.


title: Pandemic Influenza Vaccines

text: Since their compositions remain uncertain, universal pandemic vaccines are yet to be created. They would aim to protect globally against pandemic influenza viruses that have not yet evolved. Thus they differ from seasonal vaccines to influenza virus, which are updated annually in spring to incorporate the latest circulating viruses, and are then produced and delivered before the peak influenza season starts in late fall and winter. The efficacy of seasonal vaccines is linked to their ability to induce virus-neutralizing antibodies, which provide subtype-specific protection against influenza A viruses. If pandemic vaccines were designed to resemble current vaccines in terms of composition and mode of action, they would have to be developed, tested, and mass-produced after the onset of a pandemic, once the causative virus had been identified. The logistic problems of generating a pandemic vaccine from scratch, conducting preclinical testing, and producing billions of doses within a few months for global distribution are enormous and may well be insurmountable. Alternatively, the scientific community could step up efforts to generate a universal vaccine against influenza A viruses that provides broadly cross-reactive protection through the induction of antibodies or T cells to conserved regions of the virus.


title: Plant-Based Oral Vaccines: Results of Human Trials

text: Vaccines consisting of transgenic plant-derived antigens offer a new strategy for development of safe, inexpensive vaccines. The vaccine antigens can be eaten with the edible part of the plant or purified from plant material. In phase 1 clinical studies of prototype potato- and corn-based vaccines, these vaccines have been safe and immunogenic without the need for a buffer or vehicle other than the plant cell. Transgenic plant technology is attractive for vaccine development because these vaccines are needle-less, stable, and easy to administer. This chapter examines some early human studies of oral transgenic plant-derived vaccines against enterotoxigenic Escherichia coli infection, norovirus, and hepatitis B.


title: Immunomodulatory Properties of Defensins and Cathelicidins

text: Host defence peptides are a conserved component of the innate immune response in all complex life forms. In humans, the major classes of host defence peptides include the α- and β-defensins and the cathelicidin, hCAP-18/LL-37. These peptides are expressed in the granules of neutrophils and by a wide variety of tissue types. They have many roles in the immune response including both indirect and direct antimicrobial activity, the ability to act as chemokines as well as induce chemokine production leading to recruitment of leukocytes to the site of infection, the promotion of wound healing and an ability to modulate adaptive immunity. It appears that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, T cells and epithelial cells. The importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides showgreater susceptibility to bacterial infections. In the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. Although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents.


title: Respiratory Infections

text: The respiratory tract is a common site of infection in cancer patients and is associated with substantial moribidity and mortality in this population. Cancer, chemotherapy, and radiation can all cause noninfectious pulmonary infiltrates and respiratory symptoms that can masquerade as a respiratory tract infection. Cancer patients are at a particular risk for infection by a wide variety of different viruses, fungi, and bacteria that can be difficult to treat. Although noninvasive diagnostics have significantly improved recently, patients with severe pneumonia and those not responding to usual therapy should be candidates for aggressive diagnostic testing and tissue sampling. Initial therapy should be carefully chosen and individually tailored to account for the individual patient’s underlying risk factors for multi-drug-resistant pathogens, viral pathogens, or fungi. Once diagnostic testing returns, therapy should be altered to appropriately narrow the spectrum of coverage.


title: Antifibrotic Roles of RAAS Blockers: Update

text: The rennin–angiotensin–aldosterone system (RAAS) has been well documented in regulating blood pressure, fluid volume, and sodium balance. Overactivity of RAAS promotes both systemic and regional glomerular capillary hypertension, which could induce hemodynamic injury to the glomerulus, leading to kidney damage and renal fibrosis via profibrotic and proinflammatory pathway. Therefore, the use of RAAS inhibitors (i.e., ACEIs, ARBs, and MRAs) as the optional therapy has been demonstrated to prevent proteinuria, and kidney fibrosis and slow the decline of renal function effectively in the process of kidney disease during the last few decades. Recently, several new components of the RAAS have been discovered, including ACE2 and the corresponding ACE2/Ang (1-7)/Mas axis, which are also present in the kidney. Besides the classic RAAS inhibitors target the angiotensin-AT1-aldosterone axis, with the expanding knowledge about RAAS, a number of potential therapeutic targets in this system is emerging. Newer agents that are more specific are being developed. The present chapter outlines the insights of the RAAS agents (classic RAAS antagonists/the new RAAS drugs), and discusses its clinical application in the combat of renal fibrosis.


title: Risk and Response to Biological Catastrophe in Lower Income Countries

text: Natural and intentional biological risks threaten human civilization, both through direct human fatality as well as follow-on effects from a collapse of the just-in-time delivery system that provides food, energy and critical supplies to communities globally. Human beings have multiple innate cognitive biases that systematically impair careful consideration of these risks. Residents of low-income countries, especially those who live in rural areas and are less dependent upon global trade, may be the most resilient communities to catastrophic risks, but low-income countries also present a heightened risk for biological catastrophe. Hotspots for the emergence of new zoonotic diseases are predominantly located in low-income countries. Crowded, poorly supplied healthcare facilities in low-income countries provide an optimal environment for new pathogens to transmit to a next host and adapt for more efficient person-to-person transmission. Strategies to address these risks include overcoming our natural biases and recognizing the importance of these risks, avoiding an over-reliance on developing specific biological countermeasures, developing generalized social and behavioral responses and investing in resilience.


title: One Health Approach in the South East Asia Region: Opportunities and Challenges

text: The outbreaks of SARS, avian influenza, and Nipah virus in Asian countries clearly demonstrated that new highly infectious agents periodically emerge at the human–animal interface. The experiences of regional countries with prevention and control of avian influenza, SARS have reinforced the need for sustained, well-coordinated, multi-sector, multi-disciplinary, community-based actions to address emerging disease threats. ‘One Health’ is a cost-effective, sustainable, and practical approach to find solutions for problems which need holistic, multidisciplinary approaches, particularly in resource-constrained countries. While there is a growing recognition of One Health, it has to be translated from concept into actions through country level activities that are relevant for specific situations.


title: A Method to Rapidly Induce Organelle-Specific Molecular Activities and Membrane Tethering

text: In this chapter we describe a technique for rapid protein targeting to individual intracellular organelles. This method enables a real-time imaging-based study of cellular behavior in response to controlled induction of signaling events in a specifically targeted cellular compartment. We provide rationales and a step-by-step protocol for probe design and imaging of protein targeting along with two different applications of this technique. One application involves organelle-specific activation of small GTPases, while the other application involves membrane tethering of two different organelles. In the former case, we activate Rac1 and Ras at distinct intracellular locations in order to study compartmentalization of their signaling pathways, and in the latter example, we induce membrane tethering of the endoplasmic reticulum and mitochondria in order to study organelle–organelle communication. The described technique allows to rapidly perturb molecular activities and organelle–organelle communications at precise locations with specified timing and represents a powerful strategy to dissect spatiotemporally complex biological processes.


title: Natural Withanolides in the Treatment of Chronic Diseases

text: Withanolides, and in particular extracts from Withania somnifera, have been used for over 3,000 years in traditional Ayurvedic and Unani Indian medical systems as well as within several other Asian countries. Traditionally, the extracts were ascribed a wide range of pharmacologic properties with corresponding medical uses, including adaptogenic, diuretic, anti-inflammatory, sedative/anxiolytic, cytotoxic, antitussive, and immunomodulatory. Since the discovery of the archetype withaferin A in 1965, approximately 900 of these naturally occurring, polyoxygenated steroidal lactones with 28-carbon ergostane skeletons have been discovered across 24 diverse structural types. Subsequently, extensive pharmacologic research has identified multiple mechanisms of action across key inflammatory pathways. In this chapter we identify and describe the major withanolides with anti-inflammatory properties, illustrate their role within essential and supportive inflammatory pathways (including NF-κB, JAK/STAT, AP-1, PPARγ, Hsp90 Nrf2, and HIF-1), and then discuss the clinical application of these withanolides in inflammation-mediated chronic diseases (including arthritis, autoimmune, cancer, neurodegenerative, and neurobehavioral). These naturally derived compounds exhibit remarkable biologic activity across these complex disease processes, while showing minimal adverse effects. As novel compounds and analogs continue to be discovered, characterized, and clinically evaluated, the interest in withanolides as a novel therapeutic only continues to grow.


title: Operationalizing One Health: Stone Mountain and Beyond

text: Although the interconnection of humans, animals, and ecosystems has been recognized historically, increasing specialization of professionals in the twentieth century led to decreased communication and collaboration among sectors. In early 2000, a One Health vision of global interconnectedness began gaining in popularity and a series of meetings were held extolling the One Health vision. However, by 2009, detractors were claiming that the One Health approach was indeed all vision and no action. In response to this, international organizations sponsored a carefully planned and structured meeting to construct a way forward that would lead to tangible outcomes. The Stone Mountain meeting, Operationalizing “One Health”: A Policy Perspective—Taking Stock and Shaping an Implementation Roadmap led to the formation of seven multi-national work groups with defined timelines and outputs. The process has garnered increasing participation and support, and the work groups are on track to demonstrate the value added of a One Health approach.


title: Modulating cGMP to Treat Lung Diseases

text: Background: Nitric oxide (NO) is constitutively produced in the lung by NO-synthases. The main cellular sources of lung NO production are the vascular endothelium and the airway epithelia (Bohle et al. 2000; German et al. 2000; Ide et al. 1999). Local NO production contributes to regulation of pulmonary perfusion depending on alveolar ventilation to assure optimized ventilation/perfusion distribution (Grimminger et al. 1995). NO-synthase activity is regulated on transcriptional and post-translational redox-based modulation level. The common signaling pathway of endogenous vasodilators, such as nitric oxide, prostaglandins, and natriuretic peptides, engage cyclic nucleotides (cAMP and cGMP). These second messengers are mainly produced by activation of adenylate- and guanylate-cyclases, both membrane-bound and soluble (Beavo 1995). Phosphodiesterases (PDEs) represent a superfamily of enzymes, with PDE1 through PDE11 being currently known, that inactivate cyclic AMP and cyclic GMP, with different tissue distribution and substrate specificities (Ahn et al. 1991; Von Euler and Liljestrand. 1946). Because of stabilization of these second messengers, PDE inhibitors differentially regulate levels of cAMP and/or cGMP, depending on their selectivity profile. Recently, direct activators and stimulators of the sGC have been suggested as new therapeutic tools for the treatment of lung vascular disorders that might have even higher potency than PDE inhibitors or exogenously applied NO.