By D. Gunnar. Bloomfield College.
Gene therapy with plasmid vectors has been used to attempt to treat cystic ﬁbrosis (see Chapter 3) and cancer in humans (see Chapter 10) buy 500mg sumycin free shipping. A major advantage of using nonviral vectors is the lack of risk of generating a wild-type virus via recombination sumycin 500 mg on-line. In addi- tion buy 500 mg sumycin with amex, episomal plasmids do not pose the risk of insertional mutagenesis since they do not integrate into the chromosome. However, some plasmids can integrate into the genome particularly when a procedure is used to select clones exhibiting long- term expression. A second potential risk for nonviral vectors is that certain compounds can facilitate entry into a cell and exert a toxic effect in vivo. For example, many cationic lipids have considerable toxicity when administered at high doses to cells in vitro. It will be necessary to assess the toxicity of such compounds care- fully in vivo. Summary: Nonviral Vectors Nonviral vectors can be ampliﬁed to high copy numbers in bacterial cells as well as readily engineered to express a therapeutic gene from a mammalian promoter. These plasmids can be efﬁciently introduced into cells ex vivo and introduced some- what less efﬁciently into cells in vivo. Their major advantages are the ease of production and that they cannot recombine to generate replication-competent virus. They can, however, integrate at a low frequency into the chromosome and, therefore, do pose some risk of insertional mutagenesis. This binding can block access of transcription factors, thus inhibiting transcription of a gene. The triplex-forming oligodeoxynucleotide binds to the purine-rich strand of the double helix via Hoogsteen hydrogen bonds. Potential target sites for triplex formation are limited to regions that contain homopurine on one strand. The relatively weak binding afﬁnity and the instability of oligodeoxynu- cleotides in cells results in a transient effect. A third mechanism by which oligodeoxynucleotides can alter gene expression is to bind transcription factors, which prevents them from associating with endogenous genes. Natural antisense oligodeoxynucleotides consist of phosphodiester oligomers, are sensitive to nucleases, and have a half-life in serum of 15 to 60min. Modiﬁcations to the backbone have increased the stability of oligonucleotides to allow a pro- longed biological effect on targeted cells in vivo. Substitution of a nonbridge oxygen in the phosphodiester backbone with a sulfur molecule results in phosphorothioate nucleotides, which are resistant to nucleases. Substitution of a nonbridge oxygen with a methyl group results in methylphosphonate nucleotides. Modiﬁed oligonucleotides are stable in culture and serum and have resulted in prolonged biological effects. Use and Safety of Oligonucleotides for Gene Therapy Oligonucleotides have been administered in vivo for gene therapy. Oligonucleotides that served as a decoy for a transcription factor have been used to inhibit proliferation of smooth muscle cells in blood vessels in vivo. Antisense oligonucleotides have blocked expression of oncogenes, slowed replication in cells in vitro, and had a modest but transient effect upon growth of tumor cells in vivo. The major toxicity of oligonucleotides relates to the administration of large doses to achieve a clinical effect. Administration of high doses of phosphorothioate oligonucleotides resulted in cardiovascular toxicity and death in some primates. Oligonucleotides are unlikely to have any long-term adverse effects since they do not integrate into the chromosome. Oligonucleotides can alter gene expression in vitro and to a lesser extent in vivo. Their use for gene therapy will probably be limited to diseases where transient expression is sufﬁcient. These vectors are produced by cells that also express any proteins that are necessary for produc- ing a viral particle. A risk of all viral vectors is that they might recombine to generate replication-competent virus that could cause disease in humans. Plasmids can transfer a therapeu- tic gene into a cell, while oligonucleotides inhibit the expression of endogenous genes. Transfer of nonviral vectors into cells is inefﬁcient and the effect is gen- erally transient. Recently devel- oped lentiviral vectors do transduce nondividing cells, but there are concerns regarding the safety of these vectors. Adenoviral vectors transduce nonreplicating cells very efﬁ- ciently, although expression is short-lived. They have resulted in long-term expression, although it is unclear if they remain episomal or integrate into the chromosome in nondividing cells. Effect of the E4 region on the persistence of transgene expres- sion from adenovirus vectors. Gene therapy with recombinant adenovirus vectors: Evaluation of the immune response. Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression. Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Development and application of herpes simplex virus vectors for human gene therapy. The use of herpes simplex virus-based vectors for gene delivery to the nervous system. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Baculovirus Vectors Sandig V, Hofmann C, Steinert S, Jennings G, Schlag P, Strauss M. Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts.
More than 50% of lower limb amputations in the United States (70 buy discount sumycin online,000 each year) are due to diabetic foot ulcers buy sumycin 500mg low price. Immune System Dysfunction Immune system dysfunction often begins to occur long before a diagnosis of diabetes is made purchase sumycin 250mg with mastercard. In fact, in many cases a recurrent vaginal or skin yeast infection is the clue that leads to the detection of diabetes. Immune system problems are made worse by poor glucose control, and this puts the diabetic at risk for serious infections or complications of simple infections. Susceptibility to chronic, hidden infections in the oral cavity, blood, or respiratory tract may be a primary reason for increased risk of cardiovascular disease in diabetics. Depression and Cognitive Difficulties Depression and cognitive difﬁculties are common in diabetics. In fact, depression may begin to occur decades before the onset of type 2 diabetes, when the individual ﬁrst develops insulin insensitivity. The brain has a greater need for glucose than any other organ, and it appears that the brain cells may suffer from some degree of glucose deprivation when insulin resistance occurs. Cognitive changes begin to occur after the ﬁrst severe hypoglycemic episode in diabetics. Hypoglycemia is profoundly stressful to the brain, and if severe hypoglycemia occurs many times, signiﬁcant cognitive impairment is possible. Uncontrolled diabetes is also associated with an increased risk of developing Alzheimer’s disease. Contributors to Long-Term Complications of Diabetes The major factors contributing to the long-term complications of diabetes are listed here, followed by a brief description of each, along with coping measures: • Poor glucose control • Glycosylation of proteins (by means of an action similar to glycosylation of hemoglobin) • Intracellular accumulation of sorbitol • Increased oxidative damage • Nutrient deficiency • Elevated homocysteine levels • Hypertension • Changes in blood vessel linings Poor Glucose Control A large body of evidence indicates that good blood glucose control signiﬁcantly reduces the development of complications. Maintaining hemoglobin A1C levels near normal (less than 7%) can dramatically help reduce the risk of eye problems (up to 76%), nerve damage (up to 60%), and kidney disease (up to 56%). As described previously, glycosylation refers to the binding of glucose to proteins. The poorer the glucose control, the greater the binding of glucose molecules to proteins. Among the adverse effects of excessive glycosylation are inactivation of enzymes, inhibition of regulatory molecule binding, and formation of abnormal protein structures. As a result, the liver “thinks” there is a shortage of cholesterol in the body and continues to produce more and release it into the blood. This is one reason diabetes is almost always associated with high cholesterol levels. In addition to keeping blood glucose levels as close to ideal as possible, high intakes of antioxidants—especially vitamins C and E, ﬂavonoids, and alpha-lipoic acid (discussed later)—help to reduce glycosylation. Intracellular Accumulation of Sorbitol Sorbitol is a sugar molecule that is formed from glucose within cells. In people without diabetes, once sorbitol is formed it is quickly broken down into fructose. This conversion to fructose is critical because the intact sorbitol molecule cannot exit the cell, and if sorbitol levels continue to increase within a cell, the cell leaks small molecules such as amino acids, inositol, glutathione, niacin, vitamin C, magnesium, and potassium to maintain osmotic balance. Because these compounds function to protect cells from damage, their loss results in increased susceptibility to damage. Intracellular accumulation of sorbitol is a major factor in the development of most complications of diabetes, as evidenced by the fact that elevated sorbitol levels are found in high concentrations in the tissues commonly involved in the major diabetic complications: the lens of the eye, nerve cells, kidney cells, and the cells that line blood vessels. In addition to controlling blood glucose levels, vitamin C and ﬂavonoids such as quercetin, grape seed extract, and bilberry extract can help lower intracellular sorbitol levels. They also greatly increase the inﬂammatory process by increasing the formation of inﬂammatory mediators such as C-reactive protein. In addition to a basic supplementation program, supplementing the diet with antioxidants such as alpha-lipoic acid and flavonoid-rich extracts is often useful. Nutrient Deficiency A deﬁciency of any one of several nutrients has been shown to contribute to several chronic complications of diabetes. Nutrient supplementation has been found in studies to help diabetic patients with glucose control, to lower blood pressure, and to protect the body from diabetic complications. In general, the risk of long-term complications of diabetes is inversely proportional to micronutrient status. Sometimes the symptoms of nutrient deﬁciency can mimic closely a chronic complication of diabetes. For example, vitamin B12 deﬁciency is characterized by numbness, pins- and-needles sensations, or a burning feeling in the hands or feet—symptoms virtually identical to those of diabetic neuropathy. Although vitamin B12 supplementation has been used with some success in treating diabetic neuropathy, it is really not clear if this success is due to correction of a B12 deficiency state or the normalization of the deranged vitamin B12 metabolism seen in diabetics. High-potency multiple vitamin and mineral supplementation is critical to the management of diabetes. Supplying the diabetic with additional key nutrients improves blood glucose control and reduces the development of the major long-term complications of diabetes. Elevated Homocysteine Levels Elevated homocysteine levels are an independent risk factor for dementia, heart attack, stroke, and peripheral vascular disease. In addition, recent research has implicated elevations of homocysteine in the development of long-term complications of diabetes, especially diabetic retinopathy. Maintaining blood pressure in the normal range (120–140/80 mm Hg) can reduce the risk of heart disease and stroke by approximately 33 to 50% and reduce microvascular disease (eye, kidney, and nerve disease) by approximately 33% in patients with diabetes. Changes in Blood Vessel Linings A single layer of endothelial cells lines all blood vessels and acts as a metabolically active barrier between the components of blood and the blood vessel. These cells regulate many important aspects of blood ﬂow, coagulation and clot formation, and the formation of key regulating compounds, including those that control blood pressure. All of these factors have been shown to improve endothelial cell function and are critical in the battle against vascular disease in diabetes. What determines how strict the diet needs to be with regard to the intake of carbohydrates is based on the ability to get blood glucose measurements and A1C levels under control and achieve and maintain ideal body weight. Obviously, the poorer the control, the more the carbohydrate intake must be restricted. Initially, some people with diabetes—especially those who have poorly controlled blood glucose levels—may need to avoid meals with a total glycemic load of more than 20 (see Appendix B) and space these meals at least three hours apart. Meals with a higher glycemic load can be consumed if a natural product designed to slow gastric emptying and blunt after-meal blood glucose levels is used (these compounds are discussed later). Clinical Studies of Diet Therapy in Type 1 Diabetes Numerous clinical studies have shown impressive results in improving blood glucose control when diets high in ﬁber and low in glycemic load are followed. Clinical Studies of Diet Therapy in Type 2 Diabetes Diet can often be effective as the sole factor in treating and reversing type 2.
If responding and reacting to negative feedback does not take us further down the road to our own goal—or serve our ends discount sumycin 250mg without prescription, then there is no need to respond at all buy sumycin 250 mg on-line. And cheap sumycin 500mg free shipping, if response of any kind gets us off course, or works against us—then no response is the appropriate response. We must be sensitive to negative feedback data which advises us when we are off course, so that we can change direction and go forward. Our ship must not be tossed and rocked and perhaps sunk by every passing wave, or even a serious storm. As Prescott Lecky expressed it, "The same attitude must be maintained in spite of environ- mental changes. It keeps us from being tossed about, knocked off course, or "shaken up," by every wave or ripple in the environment. Not satisfied with over- responding to actual minor stimuli in the actual environ- ment, many of us create straw men in our imaginations, and emotionally respond to our own mental pictures. In addition to those negatives which actually exist in the en- vironment, we impose our own negatives: This or that may happen; What if such and such happens. When we worry, we form mental pictures—adverse mental pictures of what may exist in the environment, of what may hap- pen. Remember, your nervous system can- not tell the difference between a real experience and one that is vividly imagined. As far as your emotions are concerned, the proper response to worry pictures is to totally ignore them. Analyze your environment—be- come more aware of what actually exists in your environ- ment—and respond and react spontaneously to that. Then, your response will be appropriate—and you will have no time to notice or respond to a fictitious environ- ment. Your First Aid Kit Carry these thoughts with you as a sort of first aid kit: Inner disturbance, or the opposite of tranquility, is nearly always caused by over-response, a too sensitive "alarm reaction. You cure old habits of over-response, you extinguish old conditioned reflexes, when you practice delaying the habitual, automatic, and unthinking response. Use the quiet room in your mind technique both as a daily tranquilizer to tone down nervous response, and to clear your emotional mechanism of "carry-over" emotions Which would be inappropriate in a new situation. Practice Exercise: Create in your imagination a vivid mental picture of yourself sitting quietly, composed, un- moved, letting your telephone ring, as outlined earlier in this chapter. Then, in your daily activities "carry over" the same peaceful, composed, unmoved attitude by re-. Say to yourself, "I am let- ting the telephone ring" whenever you are tempted to "obey" or respond to some fear-bell or anxiety-bell. Next, use your imagination to practice non-response in various sorts of situations: See yourself sitting quietly and un- moved while an associate rants and raves. See yourself going through your daily tasks one by one, calmly, com- posed, unhurried, in spite of the pressures of a busy day. See yourself maintaining the same constant, stable course, in spite of the various "hurry-bells" and "pressure-bells" in your environment. See yourself in various situations which have in the past upset you—only now you remain "set," settled, poised—by not responding. Your Spiritual Thermostat Your physical body has a built-in thermostat, itself a servo-mechanism, which maintains your inner physical temperature at a steady 98. It is able to function properly in the environment because it does not take on itself the climate of the environment. You also have a built-in spiritual thermostat which en- ables you to maintain an emotional climate and atmo- sphere in spite of the emotional weather around you. Yet, your spiritual thermostat is just as necessary for emotional health and well-being as your physical thermostat is for physical health. When playing by himself, or with friends, or in small tournaments where the stakes are low, his play is flawless. People Who Come into Their Own in a Crisis For example, John Thomas, the record-breaking high- jumper from Boston University, often performs better in competition than in practice. The manager frequently turns down the man with the highest batting average, for a player who is known to "come through in the clutch. Many women are charming and gracious when talking with one person or a small informal group, but become tongue-tied, awkward, and dull at a formal dinner, or a big social occasion. On the other hand, I know a little lady who comes into her own only under the stimulus of a big occasion. Even her facial features seem to undergo a change and you find yourself thinking of her as a beautiful woman. There are students who do extremely well in day-to-day class work, but find their minds a blank when taking an examination. There are other students who are ordinary in class work, but do extremely well on important ex- aminations. If you react improperly, a crisis can rob you of the skill, control, and ability that you ordi- narily have to call upon. The so-called "money player" in sports, in business, or in social activities—the person who comes through in the clutch—who performs better under the stimulus of chal- lenge, is invariably the person who has learned either consciously or unconsciously to react well to crisis situa- tions. In order to perform well in a crisis we need to (1) learn certain skills under conditions where we will not be over- motivated; we need to practice without pressure. The crude inept stroke that he used to rescue himself becomes "fixed" and it is difficult for Mm to learn better ways of swimming. Because of his ineptness he may perish in a real crisis where he is re- quired to swim a long distance. Tolman, psychologist and expert on animal behavior at the University of California, says that both animals and men form "brain maps" or "cognitive maps" of the environment while they are learning. In the future, if this one way happens to be blocked, the animal becomes frustrated, and fails to discern alternative routes or detours. He de- velops a "one response," cut and dried, preconceived, and tends to lose the ability to react spontaneously to a new situation. Tolman found that if rats were permitted to learn and practice under non-crisis conditions, they later per- formed well in a crisis.