Manjistha Sengupta

Acetylcholine receptor antibody–mediated animal models of myasthenia gravis and the role of complement

Because of the failure of many promising therapeutics identified in preclinical evaluation, funding sources have established guidelines for increased rigor in animal evaluations. The myasthenia gravis (MG) community of scientists has developed guidelines for preclinical assessment for potential MG treatments. Here, we provide a focused summary of these recommendations and the role of complement in disease development in experimental models of MG.

Serum metabolomic response of myasthenia gravis patients to chronic prednisone treatment

Prednisone is often used for the treatment of autoimmune and inflammatory diseases but they suffer from variable therapeutic responses and significant adverse effects. Serum biological markers that are modulated by chronic corticosteroid use have not been identified. Myasthenia gravis is an autoimmune neuromuscular disorder caused by antibodies directed against proteins present at the post-synaptic surface of neuromuscular junction resulting in weakness. The patients with myasthenia gravis are primarily treated with prednisone. We analyzed the metabolomic profile of serum collected from patients prior to and after 12 weeks of prednisone treatment during a clinical trial. Our aim was to identify metabolites that may be treatment responsive and be evaluated in future studies as potential biomarkers of efficacy or adverse effects. Ultra-performance liquid chromatography coupled with electro-spray quadrupole time of flight mass spectrometry was used to obtain comparative metabolomic and lipidomic profile. Untargeted metabolic profiling of serum showed a clear distinction between pre- and post- treatment groups. Chronic prednisone treatment caused upregulation of membrane associated glycerophospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, 1, 2-diacyl-sn glycerol 3 phosphate and 1-Acyl-sn-glycero-3-phosphocholine. Arachidonic acid (AA) and AA derived pro-inflammatory eicosanoids such as 18-carboxy dinor leukotriene B4 and 15 hydroxyeicosatetraenoic acids were reduced. Perturbations in amino acid, carbohydrate, vitamin and lipid metabolism were observed. Chronic prednisone treatment caused increase in membrane associated glycerophospholipids, which may be associated with certain adverse effects. Decrease of AA and AA derived pro-inflammatory eicosanoids demonstrate that immunosuppression by corticosteroid is via suppression of pro-inflammatory pathways. The study identified metabolomic fingerprints that can now be validated as prednisone responsive biomarkers for the improvement in diagnostic accuracy and prediction of therapeutic outcome.

Drug Targeting and Delivery

Drug delivery is defined as mechanisms to introduce pharmaceutical compounds to human in order to achieve therapeutic effects. We have come a long way since chewing medicinal plants and inhaling soot from medicinal substance were the only form of drug delivery. These approaches lacked consistency and uniformity of drug delivery. Since then there has been a continuous effort to discover and improve drug delivery routes and drug delivery systems. Conventional drug delivery system includes drug delivery via oral route as solutions, suspensions, emulsions, and tablets. Some are delivered systemically via injections and intravenous application. Medications are applied topically as lotions and gels. Nasal route is used for drug delivery to lungs by inhalers and nebulizers. Apart from antibiotics, vaccines, and chemical compounds, modern medicine includes recombinant DNA, insulin, interferon, interleukin, erythropoietin, tissue plasminogen activator, and other peptides and macromolecules as drugs that require efficient drug delivery systems. Traditional drug delivery systems suffer from various limitations such as low bioavailability, intolerance, toxic side effects, reduced plasma half-life, higher concentration, and low efficacy. The hydrophilic drugs have difficulty in passing through the cell membrane. Systemically delivered drugs reach all the organs irrespective of the affected organ. This causes toxic side effects on the healthy cells. The drugs tend to degrade fast in the plasma so higher doses of drug are required and hence it becomes toxic with reduced efficacy and are expensive. The biological barriers exclude the drug from reaching the affected cells and tissues. Efficient drug targeting can improve drug delivery efficacy, reduce side effects, and lower treatment cost. Hence, much effort is given on the development of novel carriers that would meet the requirement of drug delivery systems. The main areas of research are to increase bioavailability of the drugs, increase plasma half-life, and target to specific organs or cells. This would result in lowering the dose, which would also lower drug-induced toxicity, protect bystander cells and organs from adverse side effects, and reduce medical expenses. In this chapter, we will discuss the biological barriers, advances in drug delivery systems, drug targeting, and their application in diseases.

Tissue Engineering and Artificial Organ

Tissue engineering is an exciting technique, which has the potential to create tissues and organs de novo. Tissue engineering was defined in 1988 as “application of the principles and methods of engineering and life sciences toward fundamental understanding of structure–function relationship in normal and pathological mammalian tissues and the development of biological substitutes for the repair or regeneration of tissue or organ function.” It was later summarized as “an interdisciplinary field which involves fundamentals of life sciences, medical sciences, and principles of material sciences, which can provide a functional substitute for damaged or diseased organ restoring, maintaining, or improving tissue function or a whole organ.” The existence of tissue engineering dates to the sixteenth century, when complex skin flaps were used to replace the nose. Initially, the field was recognized as a subfield of biomaterials. Most definitions of tissue engineering cover a broad range of applications; in practice, the term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin, and so on). It has the potential to produce a supply of immunologically tolerant “artificial” organ and tissue substitutes that can grow in the patient.

Animal Cell Culture and Cryopreservation

Cell culture is a process by which cells are grown under laboratory conditions outside their natural environment. The historical development of methods of cell culture is closely interrelated with tissue and organ culture. Animal cell culture has a long history of over 100 years, although major advancements have been accomplished in the last 30 years. It has become one of the major tools in life sciences. Almost 50 % of the biological products produced today or planned to be produced in the near future are based on animal cell culture. Therefore, there is an increasing interest in developing technologies for cultivation and maintenance of animal cells. Apart from developing new technologies for culturing and manipulating animal cells for producing biologics, researchers are also interested to look into developmental processes using animal cells as a model system. This chapter is designed to serve as a basic introduction to animal cell culture for the students and the laboratory workers who are interested to understand the key concepts and terminologies in this rapidly growing field.

An Introduction to Biotechnology

Biotechnology is multidisciplinary field which has major impact on our lives. The technology is known since years which involves working with cells or cell-derived molecules for various applications. It has wide range of uses and is termed “technology of hope” which impact human health, well being of other life forms and our environment. It has revolutionized diagnostics and therapeutics; however, the major challenges to the human beings have been threats posed by deadly virus infections as avian flu, Chikungunya, Ebola, Influenza A, SARS, West Nile, and the latest Zika virus. Personalized medicine is increasingly recognized in healthcare system. In this chapter, the readers would understand the applications of biotechnology in human health care system. It has also impacted the environment which is loaded by toxic compounds due to human industrialization and urbanization. Bioremediation process utilizes use of natural or recombinant organisms for the cleanup of environmental toxic pollutants. The development of insect and pest resistant crops and herbicide tolerant crops has greatly reduced the environmental load of toxic insecticides and pesticides. The increase in crop productivity for solving world food and feed problem is addressed in agricultural biotechnology. The technological advancements have focused on development of alternate, renewable, and sustainable energy sources for production of biofuels. Marine biotechnology explores the products which can be obtained from aquatic organisms. As with every research area, the field of biotechnology is associated with many ethical issues and unseen fears. These are important in defining laws governing the feasibility and approval for the conduct of particular research.

Plant Biotechnology and Agriculture

Agricultural biotechnology is the term used in crop and livestock improvement through biotechnology tools. Biotechnology encompasses a number of tools and elements of conventional breeding techniques, bioinformatics, microbiology, molecular genetics, biochemistry, plant physiology, and molecular biology. The biotechnological tools that are important for agricultural biotechnology include conventional plant breeding, tissue culture and micropropagation, molecular breeding or marker-assisted selection, and genetic engineering and GM crops. In this chapter, readers would learn about the role of biotechnology in crop improvement and the major applications of the field.

MicroRNA and mRNA expression associated with ectopic germinal centers in thymus of myasthenia gravis

Background A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. Methods MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. Results Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells. Conclusion Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.

Pharmacogenomics and Pharmacogenetics

With the information available about human genome and human proteome, it is now well understood that there are a lot of variations between individuals. These minor variations account for many differences like adverse drug reactions, which are responsible for many hospitalizations and casualties. The observed variable effect of drug is due to difference in sensitivity as some people need higher dose and some need lower dose to get similar therapeutic effect, but in some people drug has no therapeutic effects and in some shows strong adverse reactions. Some of these differential effects are due to environmental causes, or the individual’s ability to absorb or metabolize a drug may be altered or multiple drug interaction can occur (in people taking multiple drugs). Pharmacogenetics is the study of the roles of specific genes in these effects, whereas pharmacogenomics is the study of how an individual’s genetic makeup affects the body’s response to drugs or the personalized medicine deals with the concepts that for a particular disease, the rate of progression of the disease for each person is unique and each person responds in a unique way to drugs. In its broadest sense, personalized medicine includes the detection of disease predisposition, screening and early disease diagnosis, assessment of prognosis, pharmacogenomic measurements of drug efficacy and risk of toxic effects, and the monitoring of the illness until the final disease outcome is known.

Immunology and Medical Microbiology

We are continuously exposed to many pathogens through inhalation, ingestion, and touch. The immune system protects us from the majority of these pathogens as flatworms, bacteria, fungi, and viruses. We have also witnessed tremendous progress in the prevention and treatment of infectious diseases; still, they remain a major challenge and are responsible for major cause of death and disability worldwide. The immune system’s memory response and vaccination have resulted in complete eradication of many diseases. Our immune system is very adaptive and consists of a variety of cells and molecules, which play an active role in protecting us. It not only protects us from the outside pathogenic agents but also is also capable of recognizing the body’s own components. It recognizes them as self and does not induce response against them. It is known as self-/non-self-discrimination. Sometimes due to certain defects or other reasons when the immune system is not able to differentiate self, then it mounts an attack on self-components leading to autoimmunity. The importance of the immune system was recognized by early work of Dr. Edward Jenner and Louis Pasteur; they recognized the abilities of the immune system, and since then the system was gradually being explored and it laid the foundation of immunology. However, day-by-day microbes are also posing health risks as new strains are continuously being evolved. Many chemotherapeutic agents have been developed to control the spread and infections. However, microbes are also continuously developing the ability of their survival with emergence of new strains and properties. Antibiotic resistance is occurring with all classes of microbes posing a serious clinical problem in managing infections. The diseases like tuberculosis, cholera, and rheumatic fever, which were believed to be eradicated, have ferociously reemerged. The reemergence and new pathogenic agents might be the result of mutations in their genome and changes occurring in the environment. In this chapter, basic concept of the immune system and some of the diseases of the skin, gastrointestinal tract, nervous system, and respiratory system caused by microorganisms are discussed along with sexually transmitted diseases and characterization of pathogens.