Methods in Molecular Medicine – serie

Leading researchers and clinicians describe their state-of-the-art findings and hypotheses arising from a variety of different approaches to this group of diseases. Their approaches include clinical presentations, epidemiology, transgenic methods, and diagnostic tests via transmission electron microscopy and immunoblotting. The diseases treated range from human to animal spongiform encephalopathies and include Creutzfeldt-Jakob disease, mad cow disease, and scrapie.

An outstanding collection of state-of-the-art experimental methods for herpes virology. These procedures range from the more biological in vivo maneuvers to the purely molecular, and are described in detail by selected experts to ensure reproducibility. Most of the protocols rely on the herpes simplex virus as prototype, so that it then becomes relatively easy to extrapolate and make the necessary modifications required for application to other herpesviruses, especially members of the alpha group, such as PRV and EHV.

The polymerase chain reaction (PCR) is one of the most important molecular biological methods ever devised, with numerous applications to cli- cal molecular medicine. Since its description in 1985, PCR has undergone tremendous improvements, and many variations on the basic PCR theme have been published. With such a large volume of PCR-related literature, a clinical scientist wishing to use the technique will have a difficult task loc- ing the relevant information to implement it effectively. There is thus clearly a need for an up-to-date volume with detailed protocols to facilitate the setting up of those techniques most relevant to clinical applications. Unlike some other books on this topic, Clinical Applications of PCR includes only methods that are of direct relevance in clinical settings. The book is organized in three parts: an introductory section, a section on general methodology, and a final section with specific clinical applications. The first section covers the basic principles of PCR and is most useful to those new to molecular diagnosis. The next chapter includes useful tips for setting up a PCR laboratory. Section 2 then outlines some of the most commonly used PCR-based techniques in molecular diagnosis. Section 3 includes carefully chosen examples that represent typical applications of PCR in diverse clinical fields, encompassing hematology, oncology, genetics, and microbiology.

A much-needed collection of cutting-edge methods for the detection and discovery of new cancer markers, particularly molecular tumor markers. Each method is fully described by a hands-on expert who regularly uses it in the laboratory and has perfected it to a high degree of ready reproducibility. Special emphasis is given to statistical considerations in the analysis of tumor markers, recent discoveries in the field of serum and tissue marker proteins, cytogenetic markers, molecular markers, and markers for the early detection of cancer. The well-honed procedures detailed here will enable cancer researchers and clinical oncologists alike to realize the promise of these powerful new diagnostic strategies for the detection, therapy, and successful prevention of cancer.

Helicobacter pylori Protocols offers an outstanding collection of state-of-the-art protocols for the identification and molecular manipulation of H. pylori. The authoritative contributors supply detailed and readily reproducible protocols for the culturing of H. pylori, for the isolation and restriction endonuclease digestion of H. pylori chromosomal DNA, and for the transformation and insertional mutagenesis of H. pylori. They also provide molecular epidemiological techniques, including ribotyping, PCR-RFLP, and RAPD-PCR. These procedures have been developed by leading practitioners to solve the difficult technical problems created by the application of the powerful bacterial genetic and molecular cloning techniques to H. pylori.

Lectins have in the past been regarded by many scientists as curious proteins of uncertain structure and specificity that bind to carbohydrates of dubious significance themselves. All this is rapidly changing. The functional importance of glycosylation in cell-cell and cell-pathogen interactions, as well as intracellular events, has been recognized by the explosion of the science of glycobiology. This has been paralleled by the realization that lectins, once they have been well characterized, can be extremely useful tools for exam- ing structural changes in glycosylation and their functional consequences for human pathophysiology. Different lectins vary considerably in their degree of specificity. Some, such as wheatgerm agglutinin, have fairly broad specificity (for glucosamine or sialic acid), whereas others, such as Maackia amurensis, are specific not only for a single carbohydrate, but also for its linkage (2-3 linked sialic acid). Lectins with relatively broad specificity may be very useful as an adjunct to isolation or quantification of soluble glycoproteins, whereas lectins of known, and precise, specificity will be more useful for characterization of carbo- drate structure.We have included an appendix in Lectin Methods and Pro- cols that provides the known specificities of all lectins cited in the text.

The accurate and reliable diagnosis of transmissible diseases is the most powerful weapon available to ensure their control, and in some cases eradication. The detection of parasites in clinical cases, companion and farm animals, and in the environment is relatively easy since many of them are visible to the naked eye, and those that are not are readily detected by light microscopy. Fungal infections can similarly be determined. Bacteria are somewhat harder to detect. Although their presence can frequently be detected by light microscopy, differential diagnosis, beyond their gross morphology, is almost always impossible. However, most bacterial pathogens can be cultured in the laboratory and can be accurately identified by combinations of a series of simple tests such as morphology, staining, antibiotic sensitivity, biochemical analyses, nutrient dependence, and phage sensitivity. Viruses, however, have proved much more difficult; their size and absolute dependence of the host cell for propagation have rendered useless the methods traditionally used for other microorganisms. Until the development of tissue culture in the middle of this century, diagnosis was entirely dependent on the skill and experience of the clinician. But this was an unreliable process since many of the common virus infections exhibit similar clinical symptoms, such as coryza, exanthema, vomiting, diarrhea, neuralgia, and lethargy. Indeed many viral infections display clinical signs that are indistinguishable from bacterial or parasitic infections.

Laboratory studies in hemostasis have traditionally focused on abn- malities of platelet function or the quantitative and qualitative disorders that affect the proteins involved in blood coagulation. However, over the last 10 years there has been an explosion in our understanding of the molecular bases that underlie many of the inherited and acquired disorders of hemostasis. Many of these disorders are now routinely diagnosed and assessed by methods that involve genotypic analysis. Indeed in the late 1990s the distinction between molecular methods for research and for routine diagnosis is becoming incre- ingly blurred. The techniques and approaches that are used in hemostasis are manifold and published in isolation in a variety of publications. The aim, therefore, of this volume Hemostasis and Thrombosis Protocols is to pull together, into a single volume, the variety of techniques that are frequently used in the field of hemostasis. We have targeted this volume at laboratories who wish to move into the field of molecular hemostasis or who may already have some expe- ence in this area but wish to develop new areas of research and diagnosis. The chapters are wide-ranging and hopefully provide a broad overview of the d- fering applications in which these standard techniques can be used. Though the articles may appear relatively specific, the techniques contained within them are applicable to the study of many different disorders and we hope that they provide a series of ideas and concepts well-suited to problem solving.

Expert laboratory scientists describe in detail their methodologies for constructing ribozymes designed to elucidate the role of specific genes as key routes to the development of novel therapies for a wide variety of diseases. The authors review the many sites targeted with ribozymes in various diseases and provide specific accounts of the practical techniques required for the proper use of ribozymes in these systems. Their cutting-edge protocols demonstrate how to achieve ribozyme expression in distinct cellular systems, the preparation and use of high-efficiency ribozyme DNA or RNA delivery, and the studies required to prove the efficacy of ribozyme-mediated inhibition. Therapeutic Applications of Ribozymes contains all the technical support needed to make ribozyme technology a widely used and significant methodology.

A collection of cutting-edge techniques for detecting most of the major viruses that afflict mankind, including influenza, hepatitis, herpes, polio, mumps, HIV, and many more. The techniques are well-tested, easily reproducible, and readily employ all the new technologies - PCR, RIA, ELISA, and latex-agglutination - that have revolutionized the field. These methods not only make it possible to do the necessary analysis in hours instead of days, but can also be automated in a laboratory havng only low levels of biological containment. Frequently, the protocols for viruses causing human diseases can be adapted to similar viruses of veterinary importance. Through its state-of-the-art methods a physician can, for the first time, determine early in a viral infection which antiviral drug should be used and minimize the period of treatment to avoid unnecessary side effects.

The enormous advances in molecular biology that have been witnessed in . Not recent years have had major impacts on many areas of the biological sciences least of these has been in the field of clinical bacteriology and infectious disease . Molecular Bacteriology: Protocols and ClinicalApplications aims to provide the reader with an insight into the role that molecular methodology has to play in modern medical bacteriology. The introductory chapter ofMolecular Bacteriology: ProtocolsandCli- cal Applications offers a personal overview by a Consultant Medical Microbio- gist of the impact and future potential offered by molecular methods. The next six chapters comprise detailed protocols for a range of such methods . We believe that the use of these protocols should allow the reader to establish the various methods described in his or her own laboratory. In selecting the methods to be included in this section, we have concentrated on those that, arguably, have greatest current relevance to reference clinical bacteriology laboratories; we have deliberately chosen not to give detailed protocols for certain methods, such as multilocus enzyme electrophoresis that, in our opinion, remain the preserve of specialist la- ratories and that are not currently suited for general use. We feel that the methods included in this section will find increasing use in diagnostic laboratories and that it is important that the concepts, advantages, and limitations of each are th- oughly understood by a wide range of workers in the field .

An unprecedented collection of core PCR techniques useful for the study and diagnosis of human diseases. Cutting-edge and essential for today's diagnostic laboratories, these techniques heavily utilize nonisotopic, solution phase, and in situ amplification methods. A significant number of chapters describe applications exploiting the eggwhite sensitivity of PCR in the detection of rare or single cells, as in identifying fetal cells circulating in maternal blood, preimplantation embryo diagnosis, or finding circulating cancer cells. The book demonstrates over and over again the power of PCR - its high sensitivity, specificity, and ability to rapidly discriminate sequence variations.

A state-of-the-art collection of detailed synthetic procedures that lead to a variety of scaffolds, turn mimetics, peptide-bound replacements, and enzyme inhibitors. Topics range from unusual syntheses of amino acids to the use of a variety of linear and heterocyclic scaffolds in place of the peptide backbone. Important chemical procedures and methods include the transient protection of charged peptides as neutral prodrugs for improved blood-brain penetration and the replacement of peptide bonds with heterocyclic rings, olefins and fluoroolefins, and ketomethylenes. Synthetic protocols towards the transition-state mimics and reactive "warheads," applicable in enzyme inhibitors, are also disclosed.

G. E. Francis and Cristina Delgado have assembled a comprehensive collection of leading-edge protocols for drug targeting studies. In addition to the detailed step-by-step instruction, the protocols review the principal current targeting strategies, discuss how to build the constructs, examine theoretical considerations relevant to solid tumors, and describe experimental and clinical applications of targeted drug delivery systems. Also present are new chemical and biological approaches to the preparation of targeted biopharmaceuticals, including ribonuclease antibody chimeras, bispecific antibodies, enzyme antibody conjugates, and folate-bearing conjugates. Applications include the targeting of myocardial infarctions using liposomal systems, the use of monoclonal antibodies to target malaria and HIV-infected cells, the targeting of fusion proteins to leukemias and lymphomas, and the use of antioxidant enzyme antibody conjugates to target the pulmonary endothelium.Comprehensive and innovative, Drug Targeting: Strategies, Principles, and Applications documents in practical detail the major new chemical and biological targeting methods now being used to produce today's emerging rich array of novel diagnostic agents and promising biopharmaceuticals.

In recent years, molecular techniques have enhanced our ability to detect sexually transmitted infections and to conduct research to further our und- standing of sexually transmitted diseases. Molecular methods to quantitate pathogen load have also been shown to be useful for the management of HIV and other viral STDs. Existing laboratory manuals for the clinical microbi- ogy laboratory often do not include molecular methods for STDs. Sexually Transmitted Diseases: Methods and Protocols is intended to fill the need for a dedicated manual that covers all the fundamental aspects of molecular pro- cols for laboratory diagnosis, as well as research methodology for STDs, - cluding HIV. There are more than types of molecular techniques described in this book covering nine major sexually transmitted pathogens. Although mole- lar methods for the detection of such pathogens as Trichomonas vaginalis are available in the published literature, they have not been included since they are not yet widely used for laboratory diagnosis or research. Sexually Transmitted Diseases: Methods and Protocols is one of a series of books treating Methods Molecular Medicine, published by Humana Press.

In Hematologic Malignancies: Methods and Techniques, a panel of acknowledged experts review many of the key molecular methods used for the diagnosis and subsequent management of hematologic malignancies. These clinically relevant techniques range from routine test procedures to highly sophisticated methods currently offered only by specialized reference laboratories, and fall into five major groups: cytogenetics, polymerase chain reaction, flow cytometry, cytochemistry and immunochemistry, and apoptosis and cytokine receptors. Serving both clinical and experimental needs, Hematologic Malignancies: Methods and Techniques provides an array of powerful tools that will guide clinicians - especially hematologists, oncologists, and pathologists - to better diagnose and manage their patients with hematologic malignancies, and enable researchers to assess the anticancer effect of agents that impact cancer cells at the molecular level.

Experienced scientists describe - in an easily-followed format - their state-of-the-art techniques for evaluating antiviral compounds. The assays described include systems for investigating drugs used against herpesviruses, hepatitis viruses, human immunodeficiency viruses, human papillomaviruses, and influenza viruses. These well-tested methods range from cellular assays to some of the most up-to-date molecular approaches for identifying compounds that are active against viral enzymes and the development of viral resistance against drugs currently in use. Timely and comprehensive, Antiviral Methods and Protocols offers today's researchers in academia, clinical departments, and the pharmaceutical industry the powerful, reproducible, and novel methods needed to evaluate compounds effective against both acute and chronic infections.

The field of DNA vaccines has undergone explosive growth in the last few years. As usual, some historical precursors of this approach can be d- cerned in the scientific literature of the last decades. However, the present state of affairs appears to date from observations made discreetly in 1988 by Wolff, Malone, Felgner, and colleagues, which were described in a 1989 patent and published in 1990. Quite surprisingly, they showed that genes carried by pure plasmid DNA and injected in a saline solution, hence the epithet “naked DNA,” could be taken up and expressed by skeletal muscle cells with a low but reproducible frequency. Such a simple methodology was sure to spawn many applications. In a separate and important line of experimentation, Tang, De Vit, and Johnston announced in 1992 that it was indeed possible to obtain humoral immune responses against proteins encoded by DNA delivered to the skin by a biolistic device, which has colloquially become known as the “gene gun. ” The year 1993 saw the publication of further improvements in the me- ods of naked DNA delivery and, above all, the first demonstrations by several groups of the induction of humoral and cytotoxic immune responses to viral antigens expressed from injected plasmid DNA. In some cases, protection against challenge with the pathogen was obtained. The latter result was - questionably the touchstone of a method of vaccination worthy of the name.

Aging is an almost universal process within biological systems, one which leads to a decline in functional capacity, disease onset, and eventually death. There has been much interest in recent years to elucidate the molecular mec- nisms that underlie the aging process. Many theories have been proposed since the last century that aim to explain the causes of aging. There is no one theory that completely satisfies the phenotype of aging, but genetics and environm- tal factors play an important role in the etiology of age-related pathologies and the aging process. However, there is still much to be learned about the aging process which has been termed one of the last great frontiers in biology. De- graphic changes worldwide are leading to increased average life expectancies within our populations. These changes in population characteristics will impact upon the economies of the supporting society, with increasing healthcare and infrastructural costs arising from the prevalence of age-related pathologies and other physical disabilities associated with advancing years. Many researchers worldwide are working in the attempt to identify key cellular processes through which it might one day be possible to slow down the aging process and thus increase the health span of humans. Numerous research projects—from the cellular through to tissue, organ, and whole organism studies—are currently underway to investigate the mul- factorial aging process.

Over one hundred well-respected scientists comprehensively review every aspect of the biology underlying ovarian cancer and present a remarkably detailed collection of easy-to-follow methods for illuminating basic mechanisms underlying all forms of cancer. Here today's investigators will find the broadest imaginable array of molecular biology techniques specially designed to determine not only tumor genetics, expression, and protein function, but also to elucidate the genetic mechanisms by which gene and immunotherapies may be perfected. Versatile and comprehensive, Ovarian Cancer Methods and Protocols constitutes a veritable treasury of well-proven expert methods that will help basic scientists and clinical researchers alike master the powerful technologies required for effective cancer research today.

Leading clinical and laboratory scientists describe cutting-edge methods for examining the mechanisms of cellular resistance to anticancer cytotoxics in human tumors. The protocols contain detailed instructions and extensive troubleshooting tips that allow researchers effectively to study a wide variety of drug resistance mechanisms, including aspects of drug-induced cell death, drug uptake/efflux, drug metabolism, and DNA repair. Each method is designed to help identify the correlation between molecular and biochemical data and the clinical responses of the patient. Cytotoxic Drug Resistance Mechanisms illuminates all the clinically relevant mechanisms used as markers of the biological response to anticancer biotherapeutics today.

Leading experts comprehensively review all aspects of the delivery of therapeutic molecules to cells using electrical impulses - i.e., the extraordinarily promising new areas of electrogenetherapy, electrochemotherapy, and transdermal drug delivery. Their survey ranges from outlines of the basic physical principles that govern cell permeabilization by pulsed electric fields, to descriptions of the current state-of-the-art in instrumentation and electrodes, to summaries of preclinical and clinical trial results. The authors focus on drug, gene, and transdermal delivery techniques, providing detailed examples of drug delivery using electric fields from a variety of pulse generators and electrodes. Comprehensive and authoritative, Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery: Electrically Mediated Delivery of Molecules to Cells provides entre to an immensely practical set of in vivo electroporation techniques, including electrogenetherapy, electrochemotherapy, and transdermal drug delivery - techniques holding great promise for all those working toward better therapies for cancer, metabolic diseases, and vaccination.

An outstanding collection of detailed, state-of-the-art techniques for studying many aspects of nervous system cell biology. The methods span a multidisciplinary range of cellular and molecular approaches to both normal and injured nervous system function, and particularly to neurodegenerative processes. Included are basic RT-PCR techniques, cell culture systems, second-messenger signaling methods, and patch-clamp techniques. These powerful techniques will illuminate nervous system function, injury, degeneration, and the repair/regenerative process.

Over the past 20 years, technological advances in molecular biology have proven invaluable to the understanding of the pathogenesis of human cancer. The application of molecular technology to the study of cancer has not only led to advances in tumor diagnosis, but has also provided markers for the assessment of prognosis and disease progression. The aim of Molecular Ana- sis of Cancer is to provide a comprehensive collection of the most up-to-date techniques for the detection of molecular changes in human cancer. Leading researchers in the field have contributed chapters detailing practical pro- dures for a wide range of state-of-the-art techniques. Molecular Analysis of Cancer includes chapters describing techniques for the identification of chromosomal abnormalities and comprising: fluor- cent in situ hybridization (FISH), spectral karyotyping (SKY), comparative genomic hybridization (CGH), and microsatellite analysis. FISH has a pro- nent role in the molecular analysis of cancer and can be used for the detection of numerical and structural chromosomal abnormalities. The recently described SKY, in which all human metaphase chromosomes are visualized in specific colors, allows for the definition of all chromosomal rearrangements and marker chromosomes in a tumor cell. Protocols for the detection of chromosomal re- rangements by PCR and RT-PCR are described, as well as the technique of DNA fingerprinting, a powerful tool for studying somatic genetic alterations in tumorigenesis.

Asthma has rapidly become one of the most common chronic illnesses of the Western world, and its prevalence continues to rise, with the proportion of patients with more severe diseases also increasing. Faced with this problem, more researchers are focusing on the causes, mechanisms, and pathophysiology of asthma. The major hopes are that more effective drugs will become available and that preventive measures can be instituted. Increasingly, molecular and cell bi- ogy approaches are being used to characterize and understand the mechanisms of the inflammatory process that is typical of the asthmatic airway. This volume on Asthma: Mechanisms and Protocols in the Methods in Molecular Medicineseries provides an overview of the molecular mechanisms involved in asthma by p- viding extensive protocols that are being used in asthma research. Briefly, it c- ers details of methods for obtaining cells from the airways, analysis of gene and protein expression in the limited clinical samples from asthmatic airways, use of molecular and cellular tools for studying cytokine expression and release, studies of asthma-related genes and genetic polymorphisms, and understanding the - fects of asthma treatments. With such coverage, the volume ties in several dis- plines, including allergy and immunology, cell biology, pharmacology, and histology. We have continued in the spirit of the series to provide a bench book for day-to-day use.

The term "electrophoresis" was first used by Michaelis in 1909, to - scribe the migration of colloids in an electric field. The first practical elect- phoresis method was described by Tiselius in 1937. He used a U-tube filled with buffer layered on top of sample; migration could be monitored using Schlieren optics. In zone electrophoresis, the U-tube was replaced by paper, a support material employed simply to prevent or minimize diffusion of ions, so that ions applied in a narrow strip to the paper will separate and remain as relatively discrete zones. Paper was superceded by a variety of other media, - cluding cellulose acetate, hydrolyzed starch (starch gel), agarose, and polyacry- mide. The latter, in addition to being a support medium, has size-sieving properties. From the basic zone electrophoresis, other means of separation have been dev- oped. These include, isoelectric focusing, isotachophoresis, density gradient el- trophoresis, and various forms of immunoelectrophoresis. In some ways Capillary Electrophoresis (CE) has gone full circle back to the original method of Tiselius.In its simplest form, separations occur in a buffer solution within a glass (fused silica) tube and detection occurs as sample moves past an optical window. CE has rapidly developed into a technique that rivals HPLC in its versatility. All the classical electrophoretic separations-zone, IEF, and isotachophoresis-have their counterparts in CE. Excitingly so, and - thoritatively treated in Clinical Applications of Capillary Electrophoresis.

Research into gluten sensitivity has never been more popular nor more exciting. Thus a call for a new book, Celiac Disease: Methods and Protocols, devoted entirely to techniques and technology seemed a most appropriate undertaking. I am therefore grateful to Professor J. M. Walker for inviting me to complete this task for Humana Press. To do this would have been imp- sible without the contributions of friends and colleagues from around the world who have devoted so much interest to the project. It has also been necessary for them to master the unique chapter-writing skills required of every ma- script published in this series of laboratory monographs. With regard to gluten sensitivity we are in a period of great change, occasioned by the introduction of reproducible methods for cloning lymp- cytes, the application of physical methods to identify gluten sequences as T-cell antigens, the study of peptide responses in vitro and in vivo by either jejunal or rectal challenge, elucidating the locations of other genes concerned in pathogenesis, or the use of elegant immunohistocytochemical and mRNA probing techniques for analyzing the finer points of the mucosal inflam- tory response to gluten.

The era of molecular pathology has arrived. From its promising beg- nings in research laboratories, the field has grown, and continues to grow, to become a vital part of the care of an ever-increasing number of patients. Because of its recent emergence from the research taboratory, many molecuIar pathology protocols we still to be found in the primary litcramre, and have not appeared in a text. MO~PCU~Q~ Padhoiogy Protocob contains la- ratory protocoIs that have been developed by many of the authors for use in clinical molecular pathology laboratories and describe in detail Row to perform these assays. This book is therefore intended for clinical laboratory use by medical technologists and pathologists. It will dso be of interest to research workers who are performing these assays. In its broadest meaning, pathology is the study of disease, and therefore it follows that any disease for which the molecular basis is understood would be suitable as a topic for inclusion in this work. When seiecting protocols, it was necessary to place limits on the number of chapters that could be feasibly presented in a single work. Those protoculs that were selected are performed more frequently, or have achieved recognition as having important diagnostic utility in contemporary practice. A decision was made to exclude inherited genetic diseases with certain exceptions, such as those diseases that are associated with thrombotic states and are part of the traditional dumain of pathology.

This book is about melanoma—its biology, immunology, and pathology, as well as the initial use of powerful genomic tools to study its fundamental mole- lar and genetic characteristics. The study of cancer will be profoundly impacted by the Human Genome Project. I would like to discuss some of these changes. The first draft of the human genome sequence was announced in June 2000, and we have just scratched the surface of the changes it will engender in medicine. A relevant question is what are the long-term effects of the Human Genome Project for medicine? I would argue that there are three, and each of these three point toward the view that systems biology will dominate biology and medicine of the 21st century. First, the Human Genome Project introduced a new type of s- ence—discovery science. Discovery science takes a biological system (e. g. , the genome) and defines all of its elements (e. g. , the sequences of the 24 human ch- mosomes). Thus, it creates a rich infrastructure from which the classical hypo- esis-driven science can be done more effectively. The effective integration of discovery- and hypothesis-driven science is a key for systems approaches to bi- ogy and medicine. Second, the Human Genome Project has provided a “periodic table of life.