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By T. Karlen. Pittsburg State University.

Diagnostic Value: Glucose is often a part of a labora- Pathologic Changes: Severe kidney damage can 25 quality toradol 10 mg,43 10 mg toradol overnight delivery,73 tory panel even though pathologic changes in lead to increased creatinine levels trusted 10 mg toradol, especially if the 36 birds are seldom detected generic toradol 10mg on line. Juve- A rise in plasma glucose concentration starts during nile budgerigars were found to have higher concen- the scotophase, reaching peak values early during the trations than adults. Afternoon plasma glucose concentra- Pathologic Changes: Increased plasma inorganic tions in birds that are fed early during the photophase phosphate levels can be seen in some cases of severe are significantly higher when compared to fasted 2,36,73 kidney damage due to vitamin D hypervitami- birds. For example, increases occur after meals, decreased plasma inorganic phosphate levels may with excitement or stress or because of decreased occur from hypovitaminosis D (calcium level also glucose usage (diabetes mellitus). Iron is transported should not be used because they interfere with the in the plasma attached to a β-1-globulin known as formation of the phosphomolybdate complex. A recent report shows a failure to correlate serum iron levels with liver biopsy and subsequent Method: Most assays for inorganic phosphate rely on toxicologic analysis for iron. It is a major constituent of bone and a vital nificantly lower values than their free-ranging coun- cellular component, playing important roles in the terparts. Very little data different standards are used (eg, human, bovine, from birds is available. Because it is impossible to have a have little importance in diagnosing hemochroma- 74,75 species-specific standard for all species presented to tosis, but insufficient research has been performed. This enzyme functions in compensated and already calibrated in scales ex- the digestion of fat in the diet. For ters yielded higher values than the biuret method, diagnostic purposes, a blood sample from a repre- with the temperature-compensated instrument be- sentative of the same species should be included for ing consistently higher in readings than the non- comparison. Temperature stress (hypothermia or sults from the refractometer and the biuret methods hyperthermia) is associated with nitrogen loss, in- may not be possible in some species. Hypoproteinemia can reflect reduced synthesis Physiology: Most plasma proteins, with the excep- caused by chronic hepatopathies, malabsorption tion of immunoglobulins and protein hormones, are caused by chronic enteropathies (enteritis, tumors, synthesized in the liver. They form the basis of organ parasitism), increased loss caused by proteinuria due and tissue structure, operate as catalysts (enzymes) to renal disease, blood loss and malignant tumors in biochemical reactions, are regulators (hormones) (rarely seen in birds) or starvation and malnutrition. The biological activity of tious diseases that stimulate production synthesis of proteins for these various functions is dependent gamma globulin. The proteins are the yolk precursors (vitel- Electrophoresis logenin and lipoproteins), which are synthesized in Sample: Serum is most commonly used for protein the liver and transported via the plasma to the ovary electrophoresis in mammals, so fibrinogen is not in- where they are incorporated in the oocyte. Hemolysis will affect electro- phoresis results, and heparinized plasma is often Diagnostic Value: Total protein is often used as an 50,54,72 used to prevent this problem. Determi- nation of plasma protein concentrations may be of Method: Electrophoresis is used to separate differ- value in diagnosing gastrointestinal, hepatic or renal ent types of plasma proteins, making it possible to diseases. Furthermore, plasma proteins will be ab- determine their relative proportion in a particular normal in infectious diseases that cause a stimulation sample. Although determination of supported on a specific matrix, is placed in an electri- plasma proteins seldom leads to a specific diagnosis cal field, causing the different protein fractions to (eg, in the case of monoclonal gammopathies), it will migrate at varying speeds toward the anode based on help the clinician to evaluate the severity and pro- their relative charge. The length and height of each peak dietary protein, temperature stress, state of hydra- ithin the pattern indicates the relative amount of a tion, hemorrhage and inflammation. Some immunoglobulins, including IgM and IgA, also migrate in the β-globulin range. The γ-globu- Sample: Serum and lipemic specimens should be lin fraction is mainly composed of immunoglobulins 66 warmed to 37°C and vigorously mixed prior to analy- (IgA, IgM, IgE and IgG). Diagnostic Value: In healthy birds the albumin frac- Method: Usually, triglycerides are enzymatically de- tion is the largest protein fraction. Often albumin concentrations are decreased in these situ- Physiology: Triglycerides are the major storage form ations. The combined effect of these changes is a of lipids, and are a major energy source. Examples of diseases with a decrease in the A/G ratio are egg-related peritonitis, and Diagnostic Value: Triglyceride values have been in- chronic infectious diseases such as aspergillosis, psit- sufficiently evaluated in birds. Serum or plasma protein electrophoresis can be used to monitor response to treatment. When the bird Physiologic Influence: Triglyceride levels may vary responds favorably, an increase in the albumin con- based on climate, hormone influence, diet and gen- centration and a decrease in the globulin concentra- der. Increases may occur during starvation, particu- tion can be observed, which leads to normalization of larly in obese birds. In birds with liver failure, extremely shown to elevate triglyceride concentrations in some species. Gastrointestinal Pathologic Changes: Egg-related peritonitis has and renal diseases can also lead to severe hypoprote- been associated with high concentrations of trigly- inemia. Because triglyceride values are Physiologic Influence: Physiologic factors that may determined based on enzymatically released glycerol, change the protein concentration and therefore affect these values may be falsely elevated after exercise or protein electrophoresis results include gender, age, following any event that causes increased levels of blood glycerol (eg, catching birds in an aviary). However, if refer- Method: Both indirect methods (based on prelimi- ence intervals are available, hyperuricemia is a good nary hydrolysis of urea with urease) and direct meth- indicator of renal disease. Normal uric acid concen- ods (based on variations in the thiazide reaction) are trations do not guarantee that the kidneys are used for urea determination. Juvenile Physiology: In the liver, protein breakdown to amino birds have lower concentrations than adults. In mately 50% lower uric acid concentrations than do dehydrated birds, nearly all of the filtered urea is carnivorous birds. Decreased filtration may occur from ever, recent investigations have shown good correla- hypovitaminosis A-induced damage to renal epi- tion between increased plasma urea concentrations thelial cells, dehydration, intoxications or from some and renal disease in pigeons. If a toenail clip is used Physiologic Influence: Physiologic conditions are for blood collection and urates from the droppings known to change urea concentrations in mammals, contaminate the sample, the uric acid levels may be but similar effects have not been documented in falsely elevated. If the blood uric acid concentration exceeds its solu- Pathologic Changes: High urea plasma levels can bility it will be deposited in different locations in the occur in all conditions that cause low urine flow, such body. High plasma or serum concentrations of uric as dehydration or bilateral ureteral obstruction. Hypervitaminosis D -induced renal dam-3 Method: Both wet and dry chemistry systems use age is frequently associated with gout and extremely oxidation of urates by uricase as a detection method. This problem is particularly Most uricase methods are extremely specific and only common in macaws. This has been described for a few structural analogues to uric acid will interfere 2,25,43 aminoglycosides (gentamicin), and allopurinol in with the test. Physiology: In birds, uric acid is the major product of Hypouricemia is much less common in birds than the catabolism of nitrogen. Approxi- reduced synthesis of uric acid has been suggested as mately 90% of blood uric acid is eliminated by secre- one etiology. Only 50% of the healthy avian kidney is actually used for excreting protein waste, providing a large functional reserve. Sodium and chloride together represent the ma- by severe tissue damage, reduced potassium excre- jority of the osmotically active constituents of tion by diseased kidneys,25,73 adrenal disease73 or be- plasma. Physiologic Influence: In budgerigars, no gender or Hypokalemia may be caused by decreased potassium other physiologic variables have been observed. Sodium Potassium Sample: Either heparinized plasma or serum is ap- Sample: Either heparinized plasma or serum is ap- propriate for sodium assays. Electrolyte concen- electrode methods are used, whole blood is also an trations are different between serum and plasma. Differences in the electrolyte con- Hyperlipemia and hyperproteinemia will cause centrations in serum and plasma must be considered falsely low potassium levels by a mechanism similar when interpreting results. Hemolysis will elevate the plasma concentration photometry or electrochemically with a sodium ion- of potassium (500 to 700%). The last two systems are most tions were found to rapidly decline in pigeon and 30,68 commonly used. Hyperproteinemia and hyper- tracellular fluid and is primarily responsible for de- lipemia will result in falsely low potassium levels termining the volume of the extracellular fluid and caused by a decreased aqueous fraction of the total its osmotic pressure. The amount of sodium in the body is regulated adsorption spectrophotometry, flame emission spec- by the kidney. In addition, many avian species have trophotometry or electrochemically with a sodium a specialized nasal gland (salt or supraorbital gland) ion-selective electrode. The last two systems are most that is able to secrete large quantities of sodium in commonly used. The other 98% is kept fresh water for a period of time the gland shrinks so within the cells by “potassium pumps” in the cell that when returned to salt water the birds can no membranes. This mechanism of decreasing sodium concentration in the serum and Diagnostic Value: Alternatives in potassium ho- urine of birds is mediated by a pituitary-adrenal meostasis have serious consequences. Many potassium Diagnostic Value: Abnormal sodium levels that are abnormalities are the result of hemolytic samples. If they do occur, they are good indicators of a Physiologic Influence: High amounts of potassium pathologic situation. Method: An expensive blood gas instrument is neces- Pathologic Changes: Hypernatremia can occur from sary. Diagnostic Value: Clinical significance in compan- ion birds has not been thoroughly investigated. It may plasma pH) has been reported in some birds with also be caused by over-hydration as in psychogenic renal disease. Other Tests Total Carbon Dioxide Content (Bicarbonate) Delta-Aminolevulinic Acid Dehydratase Method: Heparinized plasma or serum can be used. Method: Plasma or serum can be used to measure Bicarbonate levels are determined by mixing the delta-aminolevulinic acid dehydratase colorimetrically. Most of the carbon dioxide Diagnostic Value: Delta-aminolevulinic acid dehy- produced is derived from bicarbonate, but a small dratase can be used to detect lead intoxication, and amount is generated from dissolved carbonic and decreased plasma activity is pathologic. Diagnostic Value: Bicarbonate levels are useful for Acid Phosphatase establishing whether or not acidosis or alkalosis is This enzyme consists of a number of isoenzymes in a present and, if so, how severe it is. The activity is much lower than Pathologic Changes: Increases are mainly due to that of alkaline phosphatase. Ovulation has been metabolic alkalosis and decreases due to metabolic shown to increase acid phosphatase activities. Copper Method: Atomic adsorption spectrophotometry after Reference Values for Adult Budgerigars: 21 to 26 direct dilution is the method of choice for determining mmol/l. In postmortem specimens, copper concen- samples on ice to stop the metabolism of the erythro- tration in the liver provides the best diagnostic sam- cytes. In birds, the effect of estrogens on copper Many factors can influence the color of avian urine. It can change with the ingestion of water-soluble vitamins (especially Vitamin B), the amount of uric Pathologic Changes: Copper intoxications will in- acid and feces mixed with the urine, the specific crease the serum level. Plasma Dye Clearance Test In many animal species, the hepatic uptake and The white crystalline portion of the urine in birds is excretion of different organic dyes injected intrave- seldom evaluated except for color. Pathologic Changes: Lead intoxication in some species may result in chocolate milk-colored urine In contrast, Bromsulphalein must be injected with and urates. This hemoglobinuria is common and nor- care, because perivascular injection causes severe mal for some nervous birds.

The practitio- ners of clinical forensic medicine have been given many different names throughout the years cheap toradol 10 mg without a prescription, but the term forensic physician has become more widely accepted buy 10 mg toradol with mastercard. In broad terms toradol 10 mg with mastercard, a forensic pathologist generally does not deal with living individuals purchase toradol 10mg without prescription, and a forensic physician generally does not deal with the deceased. However, worldwide there are doctors who are involved in both the clinical and the pathological aspects of forensic medicine. There are many areas where both clinical and pathological aspects of forensic medicine over- lap, and this is reflected in the history and development of the specialty as a whole and its current practice. The forensic physician must also present the information orally to a court or other tribunal or forum. This table illustrates the role of forensic physicians in the United Kingdom; roles vary according to geographic location. Police surgeon, forensic medical officer, and forensic medical examiner are examples of other names or titles used to describe those who practice in the clinical forensic medicine specialty, but such names refer more to the appointed role than to the work done. Table 1 illustrates the variety of functions a forensic physician may be asked to undertake. Some clinical forensic medical practitio- ners may perform only some of these roles, whereas others may play a more History and Development 3 extended role, depending on geographic location (in terms of country and state), local statute, and judicial systems. Forensic physicians must have a good knowl- edge of medical jurisprudence, which can be defined as the application of medi- cal science to the law within their own jurisdiction. The extent and range of the role of a forensic physician is variable; many may limit themselves to specific aspects of clinical forensic medicine, for example, sexual assault or child abuse. Currently, the role and scope of the specialty of clinical forensic medicine glo- bally are ill defined, unlike other well-established medical specialties, such as gastroenterology or cardiology. In many cases, doctors who are practicing clini- cal forensic medicine or medical jurisprudence may only take on these func- tions as subspecialties within their own general workload. Pediatricians, emergency medicine specialists, primary care physicians, psychiatrists, gyne- cologists, and genitourinary medicine specialists often have part-time roles as forensic physicians. The specific English terms forensic medicine and medical jurisprudence (also referred to as juridical medicine) date back to the early 19th century. In 1840, Thomas Stuart Traill (2), referring to the connection between medicine and legislation, stated that: “It is known in Germany, the country in which it took its rise, by the name of State Medicine, in Italy and France it is termed Legal Medicine; and with us [in the United Kingdom] it is usually denomi- nated Medical Jurisprudence or Forensic Medicine. There is much dispute regarding when medical expertise in the determination of legal issues was first used. Other historical examples of the link between medicine and the law can be found throughout the world. Amundsen and Ferngren (3) concluded that forensic medicine was used by Athenian courts and other public bodies and that the testimony of physi- cians in medical matters was given particular credence, although this use of physicians as expert witnesses was “loose and ill-defined” (4), as it was in the 4 Payne-James Roman courts. The interaction between medicine and the law in these periods is undoubted, but the specific role of forensic medicine, as interpreted by historical docu- ments, is open to dispute; the degree and extent of forensic medical input acknowledged rely on the historian undertaking the assessment. Traill (2) states that: “Medical Jurisprudence as a science cannot date farther back than the 16th century. However, the Constitutio Criminalis Carolina, the code of law published and proclaimed in 1553 in Germany by Emperor Charles V, is considered to have originated legal medicine as a specialty: expert medical testimony became a requirement rather than an option in cases of murder, wounding, poisoning, hanging, drown- ing, infanticide, and abortion (1). Medicolegal autopsies were well documented in parts of Italy and Germany five centuries before the use of such procedures by English coroners. Cassar (7), for example, describes the earliest recorded Mal- tese medicolegal report (1542): medical evidence established that the male partner was incapable of sexual intercourse, and this resulted in a marriage annulment. Beck (8) identifies Fortunatus Fidelis as the earliest writer on medi- cal jurisprudence, with his De Relationibus Medicorum being published in Palermo, Italy, in 1602. Subsequently, Paulus Zacchias wrote Quaestiones Medico-Legales, described by Beck as “his great work” between 1621 and 1635. Beck also refers to the Pandects of Valentini published in Germany in 1702, which he describes as “an extensive retrospect of the opinions and deci- sions of preceding writers on legal medicine. Late 18th Century Onward Beginning in the latter part of the 18th century, several books and trea- tises were published in English concerning forensic medicine and medical History and Development 5 jurisprudence. What is remarkable is that the issues addressed by many of the authors would not be out of place in a contemporary setting. It seems odd that many of these principles are restated today as though they are new. In 1783, William Hunter (9) published an essay entitled, On the Uncer- tainty of the Signs of Murder in the Case of Bastard Children; this may be the first true forensic medicine publication from England. John Gordon Smith writes in 1821 in the preface to his own book (10): “The earliest production in this country, professing to treat of Medical Jurisprudence generaliter, was an abstract from a foreign work, comprised in a very small space. Davis (11) refers to these and to Remarks on Medical Jurispru- dence by William Dease of Dublin, as well as the Treatise on Forensic Medi- cine or Medical Jurisprudence by O. Davis considers the latter two works of poor quality, stating that the: “First original and satis- factory work” was George Male’s Epitome of Juridical or Forensic Medicine, published in 1816 (second edition, 1821). Male was a physician at Birming- ham General Hospital and is often considered the father of English medical jurisprudence. John Gordon Smith (9) stated in The Principles of Forensic Medicine Systematically Arranged and Applied to British Practice (1821) that: “Forensic Medicine—Legal, Judiciary or Juridical Medicine—and Medical Jurisprudence are synonymous terms. Beck published the first American textbook 2 years later in 1823 and a third edition (London) had been published by 1829 (8). John Gordon Smith (9) wrote that “Every medical practitioner being liable to a subpoena, should make it his business to know the relations of physi- ological and pathological principles to the facts on which he is likely to be interrogated, and likewise the principal judiciary bearings of the case. The former of these are to be found in works on Forensic Medicine; the latter in those on Jurisprudence. Personal identity Real & apparent death Identity Sudden dath Age Survivorship Sex 8. Foeticide or criminal abortion Spontaneous combustion Infanticide Death by lightning Legitimacy Death from cold 5. The first Chair of Forensic Medicine had been established in the United Kingdom in Edinburgh in 1803—the appointee being Andrew Duncan, Jr. Subse- quent nonprofessorial academic forensic medicine posts were established at Guy’s Hospital and Charing Cross Hospital, London. In 1839 and 1875, respec- tively, academic chairs of medical jurisprudence were created in Glasgow and Aberdeen (15). The relevant areas of interest to forensic medicine and medical jurispru- dence were gradually becoming better defined. Table 2 summarizes the chap- ter contents of Principles of Forensic Medicine by William Guy (16), Professor of Forensic Medicine at King’s College, London, in 1844. Much of this mate- rial is relevant to forensic physicians and forensic pathologists working today. Thus, by the end of the 19th century, a framework of forensic medicine that persists today had been established in Europe, the United Kingdom, America, and related jurisdictions. Even though medicine and law interact more frequently in cases of living individuals, forensic pathology has long been established as the academic basis for forensic medicine. It is only in the last two decades that research and academic interest in clinical forensic medi- cine have become an area of more focused research. The recent growth in awareness of abuses of human rights and civil lib- erties has directed attention to the conditions of detention of prisoners and to the application of justice to both victim and suspect. Examples of injustice and failure to observe basic human rights or rights enshrined in statute in which the input of medical professionals may be considered at least of poor quality and at worst criminally negligent have occurred and continue to occur worldwide. The death of Steve Biko in South Africa, the conviction of Carole Richardson in England, and the deaths of native Australians in prison are widely publicized instances of such problems. Reports from the European Committee for the Prevention of Torture and Inhuman and Degrading Treat- ment in the early 1990s drew attention to the problem of lack of indepen- dence of some police doctors. The conflicting needs and duties of those involved in the judicial system are clear, and it is sometimes believed that recognition of such conflicts is comparatively recent, which would be naïve and wrong. In England and Wales, the Human Rights Act 1998, whose pur- pose is to make it unlawful for any public authority to act in a manner incom- patible with a right defined by the European Convention of Human Rights, reinforces the need for doctors to be aware of those human rights issues that touch on prisoners and that doctors can influence. It is worth noting that this law was enacted almost 50 years after publication of the European Conven- tion of Human Rights and Fundamental Freedoms. The future role of the forensic physician within bodies, such as the recently established Interna- tional Criminal Court, is likely to expand. The forensic physician has several roles that may interplay when assess- ing a prisoner or someone detained by the state or other statutory body. Three medical care facets that may conflict have been identified: first, the role of medicolegal expert for a law enforcement agency; second, the role of a treat- ing doctor; and third, the examination and treatment of detainees who allege that they have been mistreated by the police during their arrest, interroga- tion, or the various stages of police custody (18). Grant (19), a police surgeon 8 Payne-James appointed to the Metropolitan Police in the East End of London just more than a century ago, records the following incident: “One night I was called to Shadwell [police] station to see a man charged with being drunk and disorderly, who had a number of wounds on the top of his head…I dressed them…and when I fin- ished he whispered ‘Doctor, you might come with me to the cell door’…I went with him. We were just passing the door of an empty cell, when a police con- stable with a mop slipped out and struck the man a blow over the head…Boiling over with indignation I hurried to the Inspector’s Office [and] told him what had occurred. Grant rightly recognized that he had moral, ethical, and medical duties to his patient, the prisoner. Grant was one of the earliest “police surgeons” in En- gland, the first Superintending Surgeon having been appointed to the Metro- politan Police Force on April 30, 1830. In 1951, the association was reconstituted as a national body under the leadership of Ralph Summers, so that improvements in the education and training for clinical forensic medicine could be made. The Association of Forensic Physicians, formerly the Associa- tion of Police Surgeons, remains the leading professional body of forensic phy- sicians worldwide, with more 1000 members. It shows how clinical forensic medicine operates in a variety of coun- tries and jurisdictions and also addresses key questions regarding how important aspects of such work, including forensic assessment of victims and investigations of police complaints and deaths in custody, are under- taken. The questionnaire responses were all from individuals who were familiar with the forensic medical issues within their own country or state, and the responses reflect practices of that time. The sample is small, but nu- merous key points emerge, which are compared to the responses from an earlier similar study in 1997 (20). In the previous edition of this book, the following comments were made about clinical forensic medicine, the itali- cized comments represent apparent changes since that last survey. There appears to be wider recognition of the interrelationship of the roles of forensic physician and forensic pathology, and, indeed, in many jurisdic- tions, both clinical and pathological aspects of forensic medicine are under- taken by the same individual. The use of general practitioners (primary care physicians) with a special interest in clinical forensic medicine is common; England, Wales, Northern Ireland, Scotland, Australasia, and the Netherlands all remain heavily dependent on such professionals. Academic appointments are being created, but these are often honorary, and until governments and states recognize the importance of the work by fully funding full-time academic posts and support these with funds for research, then the growth of the discipline will be slow. In the United Kingdom and Europe much effort has gone into trying to establish a monospecialty of legal medicine, but the process has many obstacles, laborious, and, as yet, unsuc- cessful. The Diplomas of Medical Jurisprudence and the Diploma of Forensic Medicine (Society of Apothecaries, London, England) are internationally rec- ognized qualifications with centers being developed worldwide to teach and examine them. The Mastership of Medical Jurisprudence represents the high- est qualification in the subject in the United Kingdom. Further diploma and degree courses are being established and developed in the United Kingdom but have not yet had first graduates. Monash University in Victoria, Australia, in- troduced a course leading to a Graduate Diploma in Forensic Medicine, and the Department of Forensic Medicine has also pioneered a distance-learning Internet-based continuing-education program that previously has been serial- ized in the international peer-reviewed Journal of Clinical Forensic Medicine. In addition to medical pro- fessionals, other healthcare professionals may have a direct involvement in matters of a clinical forensic medical nature, particularly when the number of medical professionals with a specific interest is limited. Undoubtedly, the multiprofessional approach can, as in all areas of medicine, have some benefits.

Four important sequences are required in cis for replication and infection in the context of gene therapy generic toradol 10 mg online. They initiate transcription at the 5¢ end cheap 10mg toradol with amex, perform polyadenylation at the 3¢ end cheap toradol 10mg visa, and inte- grate a precise viral genome into a random site of the host cell chromosome by virtue of the att sites at either end cheap toradol 10mg on line. Although integration is a highly specific process for viral sequences, integration into the host chromosome appears to be random. Subsequent studies demonstrated that inclusion of some gag sequences (the extended packaging signal) greatly increased the titer of the vector produced. It is, however, possible to remove the coding sequences from the retroviral genome and replace them with a therapeutic gene to create a retroviral vector. The deletion of viral coding sequences from the retroviral vector makes it necessary to express these genes in trans in a packaging cell line. Packaging cell lines that sta- billy express the gag, pro, pol, and env genes have been generated. The transfer of a plasmid encoding the retroviral vector sequence into packaging cell results in a retroviral particle capable of transferring genetic information into a cell (assuming appropriate tropism). However, upon transfer of the retroviral vector into a cell, infectious particles are not produced because the packaging genes necessary for syn- thesizing the viral proteins are not present. Commonly used retroviral vectors and their salient features are summarized in Table 4. The therapeutic gene is cloned into a vector using standard molecular biology techniques. To circumvent this problem, most cell lines used in animals are infected with the vector rather than transfected. This involves transfection into one packaging cell line, which produces a vector that can infect a packaging cell line with a different envelope gene. The infected packaging cell line generally contains a few copies of the retroviral vector integrated into different sites as a provirus. N2 was the first vector using an extended packaging signal that, as noted earlier, greatly increased the titer of vector produced. This mutation prevents potentially immunogenic gag peptides from being expressed on the surface of a transduced cell. In addition, it decreases the pos- sibility that a recombination event would result in replication-competent virus since the recombinant mutant would not translate the gag gene into a protein. The retroviral packaging genes gag, pro, pol, and env code for proteins that are necessary for producing a viral particle. Retroviral vectors have deleted the retroviral coding sequences and replaced them with a promoter and therapeutic gene. The retroviral vector alone cannot produce a retroviral particle because the retroviral coding sequences are not present. These packaging genes, need to be present in a packaging cell line along with the vector in order to produce a retroviral particle that can transfer genetic information into a new cell. Packaging Cells Lines Commonly used packaging cell lines are summarized in Table 4. These lines occasionally gen- erated replication-competent virus due to homologous recombination between the vector and the packaging constructs. Development of replication-competent virus is a serious concern since it leads to ongoing infection in vivo and ultimately may cause malignant transformation via insertional mutagenesis. This decreases the chance of transcriptional activation of a downstream oncogene after transduction of a cell. One strat- egy is to separate the packaging genes into two plasmids integrated into different chromosomal locations. Another strategy is to minimize homology between the vector and packaging sequences. Some packaging systems use transient transfection to produce high titers of retroviral vector for a relatively short period of time for use in animal experimentation. Recently developed packaging cell lines are of human origin and are advanta- geous. The presence of human antibodies in human serum results in rapid lysis of retroviral vectors packaged in murine cell lines. The antibodies are directed against the a-galactosyl carbohydrate moiety present on the glycoproteins of murine but not human cells. This murine carbohydrate moiety is absent from retroviral vectors that are produced by human cells, which lack the enzyme a1-3-galactosyl transferase. Human or primate-derived packaging cell lines will likely be necessary to produce retroviral vectors for in vivo administration to humans. A suitable stable packaging cell line containing both the packaging genes and the vector sequences is prepared and tested for the presence of infectious agents and replication-competent virus. This packaging cell line can then be amplified and used to produce large amounts of vector in tissue culture. Most retroviral vectors 5 6 will produce ~1 ¥ 10 to 1 ¥ 10 colony forming units (cfu)/ml, although unconcen- trated titers as high as 1 ¥ 107cfu/ml have been reported. The original vector prepa- ration can be concentrated by a variety of techniques including centrifugation and ultrafiltration. Vectors with retroviral envelope proteins are less stable to these con- centration procedures than are pseudotyped vectors with envelope proteins from other viruses. Cells that have been modified ex vivo with a retroviral vector include hematopoietic stem cells, lymphocytes, hepatocytes, fibroblasts, keratinocytes, myoblasts, endothelial cells, and smooth muscle cells. For many organs, the requirement of cellular replication for transduction poses a problem since termi- nally differentiated cells in organs are not proliferative. Thus, retroviral organ-based gene therapy approaches necessitate the induction of cell replication for in vivo transfer into cell types such as hepatocytes, endothelial cells, or smooth muscle cells. Alternatively, the use of viral vectors that do not require cellular replication could be used to transfer genes into nondividing cells in vivo. Retroviral vectors have been directly injected into malignant cells in various locations, as malignant cells are highly proliferative. Efficient in vivo delivery will likely require human or primate-derived packaging cell lines or pseudotyping to prevent complement-mediated lysis in all clinical applications of retroviral gene therapy. After transfer into a replicating cell, the expression of the retroviral vector is crit- ical to achieve a therapeutic effect. In the application of retroviral vectors for gene therapy, the relatively low levels of gene expression achieved in animals are prob- lematic. For currently selected genes used for gene therapy, the level of expression of the gene product does not need to be tightly regulated for clinical effectiveness. However, for diseases such as diabetes mellitus or thalassemia, the level of expres- sion of insulin or b-globin, respectively, requires precise control. Thus, a specific clin- ical condition may not only require a threshold level for therapeutic effectiveness but may also require a narrow window of concentration for physiological effect. There is a paucity of quantitative data in animals regarding the levels of expression per copy from different vectors, particularly in the context of organ-specific gene expression. First, current delivery systems make the experimental determi- nation of surviving transduced cells in situ difficult. Accurate determation of the copy number present in vivo is necessary since overall protein expression is a func- tion of both the number of transduced cells and the gene expression per cell. Third, the genomic inte- gration site can dramatically influence the expression level. For delivery systems that modify a small number of stem cells, such as in bone marrow stem-cell-directed gene therapy (see Chapter 7), considerable variation in expression occurs based on animal species. This variation makes it essential to quantitate expression in a large number of animals and report the average results. Thus, an improved understand- ing of the regulatory controls of gene expression from retroviral vectors remains essential for the clinical application of gene therapy in humans. Unfortunately, expression of vectors in differentiated cell types in vitro does not accurately predict expression levels that can be achieved in vivo. Retroviral vectors that alter these inhibitory sequences are expressed in vitro in embryonic carcinoma cells and may also be expressed in vivo. It is unclear, however, whether methylation per se is responsible for inactivation of the promoter or if methylation is a by-product of binding to the promoter. Retroviral vectors can include an internal promoter located immediately upstream of the therapeutic gene. These “internal promoters” can be viral promot- ers, housekeeping promoters, or organ-specific promoters. Viral promoters were components of many first-generation vectors because they are active in most cell types in vitro. This loss of function could reflect the absence of transcription factors that are essential for expression of the promoter or the presence of inhibitory proteins that terminate viral promoter activity in nonreplicating cells. Internal promoters may also comprise the ubiquitously expressed housekeeping promoters that direct the expression of proteins required by all cells. However, housekeeping genes are often expressed at relatively low levels, and their promoters have been shown to be relatively weak in vitro and in vivo in retroviral vectors constructs. Alternatively, organ-specific promoters have two major advantages: (1) allowing limited expres- sion to specific cell types or tissues and (2) directing high levels of gene expression. Muscle- or liver-specific enhancers and/or promoters, in comparison to housekeep- ing or viral promoters, direct higher levels of expression in vivo. In other studies, however, organ-specific promoters have been inactivated in vivo in transgenic mice or in a retroviral vector by the presence of adjacent retroviral sequences. It is also possible that these inhibitory sequences can decrease expression from adjacent internal promoters. The control of gene expression in vivo may be an appropriate mechanism to decrease variability in expression as well as decrease the chance that the therapeu- tic gene is overexpressed. In clinical situations, variability or overexpression would have adverse therapeutic effects. Inducible expression systems have been developed to tightly regulate expression from a retroviral vector through responsivness to an orally administered drug. However, this system requires the all- important introduction of a drug-responsive transcription factor. This is an addi- tional burden to the individual cell, which needs to receive and express two separate genes. Other factors, in addition to the choice of the promoter, can influence gene expression from a retroviral vector. For some genes and through an unknown mech- anism, the presence of a splice site dramatically increases the level of expression of the protein. Inclusion of genomic splice sites from the therapeutic gene is techni- cally difficult. Through the use of a selectable marker gene and a therapeutic gene, it is possible to eliminate cells not expressing the therapeutic gene by either in vitro or in vivo selection methods. Using these vectors, however, cells selected by virtue of expression of one gene product have a lower level of expression of the second gene product. Risks of Retroviral Vectors There are two major concerns in the use of retroviral vectors for gene therapy in humans: (1) insertional mutagenesis and (2) generation of wild-type virus.

The habit is particularly insidious because it is seldom recognized for what it is cheap toradol 10mg fast delivery. When we feel jittery cheap 10mg toradol fast delivery, or worried buy toradol 10 mg line, or anxious in thinking of the great amount of work that lies before us toradol 10 mg free shipping, the jittery feelings are not caused by the work, but by our mental attitude—which is "I ought to be able to do this all at once. When we work with this attitude, we are relaxed, we are free from the feelings of hurry and anxiety, and we are able to concentrate and think at our best. He had found, through many years of counselling, that one of the main causes of breakdown, worry, and all sorts of other personal problems, was this bad mental habit of feeling that you should be doing many things now. Just as only one grain of sand could pass through the hourglass, so could we only do one thing at a time. It is not the job, but the way we insist upon think- ing of the job that causes the trouble. Gilkey, because we form a false mental picture of our duties, obligations and responsibilities. There seem to be a dozen different things pressing in upon us at any given moment; a dozen different things to do; a dozen different problems to solve; a dozen different strains to endure. No matter how hurried or harried our existence may be, said Dr, Gilkey, this mental picture is entirely false. Even on the busiest day the crowded hours come to us one moment at a time; no matter how many problems, tasks or strains we face, they always come to us in single file, which is the only way they can come. To get a true mental picture, he suggested visualizing an hourglass, with the many grains of sand dropping one by one. This mental picture will bring emotional poise, just as the false mental picture will bring emotional unrest. Just as an electronic brain cannot give the right answer if three different problems are mixed up and fed in at the same time, neither can your own success mecha- nism. The shoemaker found that if he cut out the leather, and laid out the patterns before retiring, little elves came and actually put the shoes together for him while he was sleeping. Edison has said that each eve- ning her husband would go over in his mind those things which he hoped to accomplish the next day. Sometimes, he would make a list of the jobs he wanted to do, and problems which he hoped to solve. Bechterev said, "It happened several times when I concentrated in the evening on a subject which I had put into poetic shape, that in the morning, I had only to take my pen and the words flowed, as it were, spontaneously, I had only to polish them later. Joseph Rossman, in the Psy- chology of Invention, says, "When stumped by something, he would stretch out in his Menlo workshop and, half- dozing, get an idea from his dream mind to help him around the difficulty. Priestley dreamed three essays, complete in every detail—"The Berkshire Beast," "The Strange Outfitter," and "The Dream. He kept a number of ideas "hatching" and each night before retiring would select an "incubating idea" and "stir it up" by thinking intensely about it. Vic Pocker arrived in this country from Hungary with no money and unable to speak English. His savings were wiped out in the depression, put in 1932 he started a small welding shop of his own, which he called Steel Fabricators. Practice Exercise: In Chapter Four you learned how to induce physical and mental relaxation while resting. Con- tinue with the daily practice in relaxation and you will become more and more proficient. In the meantime, you can induce something of "that relaxed feeling," and the relaxed attitude, while going about your daily activities, if you will form the habit of mentally remembering the nice relaxed feeling that you induced. Stop occasionally during the day, it need only take a moment, and remem- ber in detail the sensations of relaxation. Sometimes forming a mental picture of yourself lying in bed, or sit- ting relaxed and limp in an easy chair helps to recall the relaxed sensations. Mentally repeating to yourself several times, "I feel more and more relaxed," also helps. You will be surprised at how much it reduces fatigue, and how much better you are able to handle situations. For by relaxing, and maintaining a relaxed attitude, you re- wove those excessive states of concern, tension and anx- iety, which interfere with the efficient operation of your creative mechanism. In time, your relaxed attitude will become a habit, and you will no longer need to consciously Practice it. Happiness is Good Medicine Happiness is native to the human mind and its physi- cal machine. We think better, perform better, feel better, and are healthier when we are happy. Kek- cheyev tested people when they were thinking pleasant and unpleasant thoughts. He found that when thinking pleasant thoughts they could see better, taste, smell and hear better, and detect finer differences in touch. Wil- liam Bates proved that eyesight improves immediately when the individual is thinking pleasant thoughts, or visu- alizing pleasant scenes. Margaret Corbett has found that memory is greatly improved, and that the mind is re- laxed, when the subject is thinking pleasant thoughts. Harvard psychologists studied the correlation between happiness and criminality and concluded that the old Dutch proverb, "Happy people are never wicked," was scientifically true. They found that a majority of criminals came from unhappy homes, had a history of unhappy human relationships. A ten-year study of frustration at Yale University brought out that much of what we call immorality and hostility to others is brought about by | our own unhappiness. Schindler has said that unhap- piness is the sole cause of all psychosomatic ills and that happiness is the only cure. It appears that in our popular thinking about happi- ness we have managed to get the cart before the horse. Happiness is not a moral issue, any more than the circula- tion of the blood is a moral issue. Happiness is simply a "state of mind in which our thinking is pleasant a good share of the time. One of the most pleasant thoughts to any human being is the thought that he is needed, that he is important enough to help and add to the happiness of some other human being. However, if we make a moral issue out of happiness and conceive of it as something to be earned as a sort of reward for being unselfish, we are very apt to feel guilty about wanting happiness. Happi- ness comes from being and acting unselfishly—as a natural accompaniment to the being and acting, not as a "pay off" or prize. If we are rewarded for being unselfish, the next logical step is to assume that the more self-abnegat- ing and miserable we make ourselves, the more happy we will be. The premise leads to the absurd conclusion that - the way to be happy is to be unhappy. If there is any moral issue involved it is on the side of happiness rather than unhappiness. It but fastens and perpetuates the trouble which occasioned it, and increases the total evil of the situation. I have found that one of the commonest causes of un- happiness among my patients is that they are attempting to live their lives on the deferred payment plan. They do not live, nor enjoy life now, but wait for some future event or occurrence. They will be happy when they get married, when they get a better job, when they get the house paid for, when they get the children through col- lege, when they have completed some task or won some victory. Riches and honors, power and pleasure, have waited on my call, nor does any earthly blessing appear] to have been wanting to my felicity. In this situation, I have diligently numbered the days of pure and genuine happiness which have fallen to my lot; they amount to fourteen. But we can, by taking thought, and making a simple decision, be happy and think pleasant thoughts a large share of the time, re- garding that multitude of little events and circumstances of daily living which now make us unhappy. To a large extent we react to petty annoyances, frustrations, and the like with grumpiness, dissatisfaction, resentment and irri- tability, purely out of habit. Much of this habitual unhappiness-reaction originated because of some event which we interpreted as a blow to our self-esteem. Even impersonal events can be interpreted, and reacted to, as affronts to our self-esteem. The bus we wanted to catch had to be late; it had to go and rain when we had planned to play golf; traffic had to get into a snarl just when we needed to catch the plane. They act like sheep—as if they were slaves, and meekly react as they are told to react. You are letting outward events and other people dictate to you how you shall feel and how you shall react. Later, I was told I could never take post-graduate courses in Germany, and that it was impossible for a young plastic surgeon to hang out his own shingle and go into business for himself in New York. I did all these things—and one of the things that helped me was that I kept reminding myself that all these "impossibles" were opinions, not facts. I not only managed to reach my goals—but I was happy in the process—even when I had to pawn my over- coat to buy medical books, and do without lunch in order to purchase cadavers. But I kept reminding myself that it was merely my opinion that this was a "catastrophe" and that life was not worth living. I not only got over it, but it turned out that it was one of the luckiest things that ever happened to me. The Attitude That Makes for Happiness It has been pointed out earlier that since man is a goal- striving being, he is functioning naturally and normally when he is oriented toward some positive goal and striving toward some desirable goal. Happiness is a symptom of normal, natural functioning and when man is functioning as a goal-striver, he tends to feel fairly happy, regardless of circumstances. He maintained an aggressive attitude, he was still goal-oriented despite his misfortune. Hollingworth has said that happi- ness requires problems, plus a mental attitude that is ready to meet distress with action toward a solution. Refuse to admit their badness; de- spise their power; ignore their presence; turn your atten- tion the other way; and so far as you yourself are con- cerned at any rate, though the facts may still exist, their evil character exists no longer. Since you make them evil or good by your own thoughts about them, it is the ruling of your thoughts which proves to be your principal con- cern. Many times I did not know from month to month where the money was coming from to pay my rent. I had a consuming desire to reach it, and a determined persistence which kept me working toward it. I related all this to the young business executive and suggested that the real cause of his unhappy feeling was not that he had lost $200,000, but that he had lost his goal; he had lost his aggressive attitude, and was yielding passively rather than reacting aggressively. Within five years he not only had more money than ever before in his life, but for the first time he was in a business that he enjoyed. Practice Exercise: Form the habit of reacting aggressively and positively toward threats and problems. Do this by practicing a positive aggressive attitude, both in actual everyday situations which come up, and also in your imagination. See yourself in your imagination taking positive, intelligent action toward solv- ing a problem or reaching a goal. See yourself reacting to threats, not by running away or evading them, but by meeting them, dealing with them, grappling with them in an aggressive and intelligent manner.