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By H. Kaelin. University of North Carolina at Greensboro. 2019.

Several studies have dem- onstrated the protective effect of breastfeeding against 11 order levitra soft online pills erectile dysfunction drugs nz. For neonates injury can often be observed buy levitra soft amex erectile dysfunction vyvanse, any ureteral injury should be born at term order 20mg levitra soft with mastercard erectile dysfunction treatment doctor, Gram-negative bacteria comprise the stented and/or repaired immediately. Escherichia or delay in therapy leads to increased complications from coli alone accounts for 80% of such infections in urinary leakage, including infected urinoma and possible neonates and young infants with other Gram-negative urosepsis [43]. Unlike the organisms carry the potential risk of being multid- gastrointestinal tract, it is usually a sterile space with rug-resistant, complicating antibiotic management an impermeable lining. The of pathogens into the urinary tract and subsequent infrequent episode of Gram-positive urosepsis in the adherence to it. Although normal voiding with inter- neonate may be caused by Group B Streptococcus, mittent urinary outflow usually clears pathogens Enterococcus or Staphylococcus (saprophyticus or within the bladder, human urine has enough nutrients aureus) species. Nelson premature infants represent a microbiologically dis- levels, and liver function. In a study of urinary tract infection in may facilitate diagnosis, but these are not specific. Similarly, a patient with otherwise unex- suddenly develop signs and symptoms of septic shock plained signs of systemic inflammation should be eval- to consider the possibility of urosepsis even in the uated for infection by history, physical examination, absence of urinary symptoms. A typical presentation includes after recent instrumentation or catheterization of the fever (temperature > 38°C or >100. A urine extremities become cool and pale, with peripheral cya- specimen obtained by catheterization or suprapubic aspi- nosis and mottling. As severe sepsis or septic shock ration must be obtained for culture before the institution develops, the first neurological signs may be confusion of antibiotic therapy. A screening renal ultrasound is producing additional signs and symptoms specific an excellent means to quickly and accurately assess the to the organ involved, including the lungs, kidneys, urinary tract in such infants. As soon as the necessary cultures have been The first priority in severe cases of urosepsis is the taken (at least two blood cultures as well as cultures initiation of basic resuscitative measures within the from urine and other appropriate body sites and flu- first 6h of presentation [9, 31, 45]. It is essential to ids), the patient should be started on broad-spectrum establish intravenous access and to administer fluids. The first-line vasopressors in this context are norepinephrine bitartrate or dopamine 11. Because norepine- The selection of initial empiric antibiotics is based phrine has little effect on cardiac output, dobutamine upon the most likely organisms involved, and the may be used concomitantly for inotropic support. A history of previous antibi- not be used as first-line therapies in septic shock. Because essary in specific circumstances as part of the initial the predominate organisms responsible for urosepsis resuscitation such as close monitoring of fluid status at all ages are Gram-negative rods, empiric therapy is particularly with regard to urine output. Patients should be say, however, that empiric treatment decisions should monitored closely for renal insufficiency secondary to be made with disregard to Gram-positive organisms, sepsis, which may require adjustment of fluid status, especially Enterococcus species. It is also impor- electrolytes, and frequent assessment of renal function tant to understand that in the context of a chapter as well as monitoring drug levels while using antibiot- addressing empiric antimicrobial recommendations ics such as aminoglycosides, or other renally excreted for urosepsis, it is implied that the treating clinician’s medications [4, 14, 15, 36, 49, 50]. The recommendations offered management of urosepsis consists of elimination of here are not necessarily appropriate for sepsis in the infectious focus or foci and initiation of appropri- general, as a number of other potential organisms ate empiric antimicrobial therapy. A list of commonly not often associated with infection of the urinary used parental antibiotics can be found in Table 11. Changes in empiric antimicrobial therapy 6 h, max 12 g per 24 h must also be considered when a patient is failing to Ceftriaxone 75 mg kg−1 per 24 h single daily dose, improve clinically within the first 24–48h of initia- max 2 g per 24 h −1 tion of therapy. Ampicillin plus gentamicin remains a reason- Cefotaxime 100–200 mg kg−1 per 24 h divided able empiric combination therapy for newborns, every 8 h, max 12 g per 24 h infants, and children with urosepsis. Nelson an expanded spectrum cephalosporin such as cefo- bacteria may respond to antibiotics to which these bac- taxime or ceftriaxone may be appropriate in addition teria appear to be resistant to in-vitro, recommended to or in place of gentamicin, especially when other therapy for such infections usually includes the use body foci such as the meninges are documented or of a carbapenem (imipenem, meropenem, ertapenem) suspected to be infected. Many experts recommend in combination with an aminoglycoside (gentamicin, use of an expanded spectrum cephalosporin in this tobramycin, or amikacin). For patients who have been the use of the dual peptide antibiotic, quinupristin/dal- hospitalized for more than 7 days and for newborn fopristin. However, due to increasing resistance to this infants born to mothers who were hospitalized for agent and the potential for infusion-related side effects, more than 7 days prior to delivery, antibiotic-resistant the use of oral or intravenous linezolid has become the Gram-negative and Gram-positive pathogens must be recommended therapy for this pathogen. Patients with these upper tract especially when such infants are not showing signs of foci of funguria should be treated with systemic therapy clinical improvement within 24–48h of initiation of that consists of amphotericin B or fluconazole. Surgical removal may be necessary An important issue pertinent to patients with urosepsis is should the fungal balls persist [45, 55, 56]. Both are very effective options that may associated, methicillin-resistant Staphylococcus aureus be used acutely to relieve the obstruction. Each of these may be urinary tract pathogens, sible, removal of urinary calculi from patients who have and each requires special antimicrobial therapy to suc- pyelonephritis is probably best delayed until the bacterial cessfully treat. The clinician treating patients with uro- load can be reduced and the patient stabilized with med- sepsis caused by one of these pathogens should work ical therapy [53]. The indications for nephrostomy tube closely with an experienced, state-of-the-art microbiol- placement include the preservation of renal function, ogy laboratory as well as consult a specialist in infec- relief of pain; and, in the most extreme circumstances, tious diseases to assist in managing these difficult the emergent drainage of a pyocalix or pyonephrosis and potentially life-threatening infections. Renal replacement therapy should be consid- cefotaxime, and cefepime as well as semisynthetic ered in patients with urosepsis who have developed renal penicillins such as ticarcillin and piperacillin. Crit Care Med 32(11 Suppl):455–465 patients, with deleterious effects on renal, cardiac, res- 3. This severe and often life-threatening and safety of recombinant human activated protein C for condition is a paradigm for the need for collaboration severe sepsis. N Engl J Med 344(10):699–709 between various specialties because the fate of the 5. Crit Care Med 32(11 Suppl):495–512 collaborative treatment involving the pediatric intensiv- 6. Bryant K, Maxfield C, Rabalais G (1999) Renal candi- ist, nephrologist, the urologist, and the infection disease diasis in neonates with candiduria. Crit Care Med 32(3):858–873 however, seeding from systemic infection is a significant 10. Intern Med 33:231–252 › Patients with obstruction or anatomic abnormalities at any 13. Sepsis associated with immunosuppressive medica- › In urosepsis it is very crucial to initiate treatment in the tions: an evidence-based review. Crit Care Med 32(11 first 6 h of presentation including fluid support, stabiliza- Suppl):578–590 tion of hemodynamic state with pressors if indicated, and 16. Pediatrics 69(4):409–412 › Renal ultrasound should be performed in cases of urosepsis 17. J Pediatr species in hospital-acquired urinary tract infections in a neo- 123:17–23 natal intensive care unit. Semin Perinatol 27:393–400 Nosocomial infections in pediatric intensive care units 24. National Nosocomial Infections Prospective multicenter surveillance study of funguria in Surveillance System.

Radiographs are indi- cated if spinal trauma or heavy met- al intoxication is suspected purchase levitra soft 20mg fast delivery impotence meme. Elec- tromyograms buy generic levitra soft 20 mg impotence group, nerve conduction ve- locities cheap levitra soft online mastercard impotence 27 years old, spinal evoked potentials and nerve or muscle biopsies are helpful in evaluating neuropathies. When avail- recumbent, and a deep pain response could not be elicited from either pelvic limb. Radiographs indicated a puncture wound through the lung (arrow) with an increased soft able, electrodiagnostic techniques tissue density (blood) in portions of the lung parenchyma. The bird was placed on are valuable in avian patients for broad-spectrum antibiotics and steroids. A deep pain response was noted five days after distinguishing between a neuropa- the initial injury, and the bird slowly improved with a complete return to normal function over a three-month period. A nerve stimulator is used to generate an M consists of insertion potentials, motor unit potentials response at two different locations along the course and spontaneous waves, which occur infrequently. The distance between the sites When the electrode is inserted into the muscle, the is divided by the latency difference in the two M intrafascicular nerve branches and muscle fibers are responses to determine the velocity with which the stimulated, creating a brief burst of electrical activ- impulse travels along the nerve (m/s). Where there is ity, which ceases immediately after the electrode a peripheral neuropathy such as demyelination, the stops moving. If the electrode is moved, insertional velocity is slow and the M responses are polyphasic activity will again be recorded. If the electrode is inserted stimulation distal to the site will produce an M re- coincidentally near a motor endplate, a low continu- sponse, while stimulation of the site proximal to the ous level of electrical activity will be recorded with an lesion will not. The H-wave reflex muscle contraction or when a motor nerve is stimu- evaluates both the afferent and efferent pathways. The M response has two peripheral site is stimulated and sensory impulses phases and represents the sum of the electrical activ- are carried to the spinal cord, where the alpha motor ity of all of the muscle fibers in that motor unit. Prolonged insertional activ- with a lower intensity than that required to cause an ity due to muscle hyperexcitability occurs six to ten H-wave production, activating the motor neuron to days following peripheral nerve injury, then gradu- generate an efferent impulse. Fibrillation potentials are mono- or wave reflexes are used in combination to evaluate biphasic and occur five to seven days following den- nerve root avulsion. These spontaneous, repetitive action poten- Signal-averaging capabilities are required for soma- tials from muscle fibers, not produced by nerve im- tosensory-evoked potentials, spinal-evoked poten- pulses, occur because of the instability of the cell tials and motor-evoked potentials. Fibrillation potentials evoked potentials correspond clinically to the increase for several weeks after denervation, then presence or absence of pain perception. They utilized to determine if failure to react to a painful stop if reinnervation occurs. This procedure is performed denervation, but may be observed with primary by stimulating a peripheral nerve and recording the myopathies. They are characterized by an initial response in the cervical spinal cord or cerebral cor- positive deflection followed by a slower negative po- tex. It is important to recognize that these evoked tential with a “dive bomber engine” auditory compo- potentials evaluate sensory, not motor nerve func- nent. A response may persist after permanent loss of potentials are generally indicative of a primary motor function. Physical examination revealed a discharge of necrotic debris from the right ear canal that contained gram-negative rods. They can discretely evaluate the sensory path- way of a focal segment of spinal cord such that loss of response cranial to a specific vertebra identifies the location of the lesion. Motor-evoked poten- tials are capable of evaluating motor function, but techniques are not well established for animal use. They are currently being evaluated for safety, effects of anesthetics and correlation with injury. They vary with head size, environment, re- straint techniques and state of consciousness of the Nutritional patient. They may be beneficial in monitoring pro- Hypovitaminosis E and Selenium Deficiency gress in response to brain lesions or injury. Auditory- evoked potentials evaluate the brainstem response Deficiencies of vitamin E and selenium have been to auditory stimuli. They may be used to assess reported to cause a wide variety of clinical signs and hearing ability and brainstem function (Figure 28. In a survey of central nervous system lesions from animals in a zoological collection, birds had a higher incidence of disease than mammals, and encephalomalacia his- tologically compatible with hypovitaminosis E was the most common lesion observed. In young birds, hypovitaminosis E may cause encephalomalacia, exudative diathe- sis or muscular dystrophy. Encepha- lomalacia results in ataxia, head tilt, circling and occasionally convulsions and is particularly common in hatch- ling budgerigars. The myositis associated with hypovitami- nosis E may cause clinical changes dif ficult to distinguish from neurologic signs. Clinical signs associated with vitamin E and sele- nium deficiencies include tremors, ataxia, incoordi- nation, abnormal head movements, reluctance to walk and recumbency. At presentation, the bird was recumbent and had stiff, nonmotile thoracic and pelvic limbs, but was bright, alert and cal signs or in birds that are found dead in their responsive. The muscle masses associated with all limbs were enclosures with no premonitory signs. His- Muscular dystrophy is characterized by light-colored topathology indicated severe, progressive, generalized muscle fi- brosis of undetermined etiology. Early histologic changes include hyaline degeneration, mi- tochondrial swelling, loss of striations and central been reported in a variety of other species including migration of the nucleus. Clinical signs include slow or incomplete eye blink due to paresis of the lower eyelid, weak jaw muscles, This deficiency has also been incriminated as the paresis of the tongue, poor digestion with passage of etiology of cockatiel paralysis syndrome. This condi- partially digested food, diminished playful activity, tion appears to occur most frequently in lutino cocka- hyperactivity, clumsiness and weak grip, low-pitched tiels infected with Giardia sp. Cockatiels and other nestling budgerigars with riboflavin deficiency (see psittacine birds showing these clinical signs have Color 48). Other signs include weakness, emaciation responded to vitamin E and selenium supplementa- in the presence of a good appetite, diarrhea, walking tion and antiprotozoal therapy. In one study, treat- on the hocks with toes curled inward and atrophy of ment of giardiasis resulted in an increase in serum leg muscles. Chicks fed a deficient diet may develop vitamin E levels from a deficiency state into the clinical signs as early as 12 days of age. There is Schwann cell proliferation sis in fledgling mocking birds fed a commercial cat and swelling, perivascular leukocytic infiltration and food has been reported. All affected birds cord and degeneration at the neuromuscular end- responded to parenteral administration of selenium plate may also be observed. Other ministration of oral or parenteral riboflavin and diet species of birds being raised at this facility were not correction; however, many of the changes are irre- affected.

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The overall survival of these patients was 58 % and it appeared to be significantly reduced in patients with multiple organ dysfunction syndrome and fluid overload generic levitra soft 20 mg on-line erectile dysfunction treatment in thane, weighing less than 10 kg or receiving stem cell transplantation [1] order levitra soft pills in toronto erectile dysfunction los angeles. They found that late initiators (>5 days) had higher mor- tality than early initiators (≤5 days) with a hazard ratio of 1 purchase levitra soft paypal erectile dysfunction treatment with viagra. Typically, in pediatric cardiac surgery neonates, in order to avoid excessive intra-abdominal pressure rise during dialysis solution infusion and to pre- vent hemodynamic instability, a “low flow” prescription of 10 ml/kg dialysate is recommended [8]. Dwell times may vary from 10 to 30 min according to the need for higher to lower solute clearances. Water ultrafiltration may be regulated by dial- ysate tonicity (provided by glucose concentration, 1. The choice of dialysis modality to be used is influenced by several factors, including the goals of dialysis, the unique advantages and disadvantages of each modality, and institutional resources. Intermittent dialy- sis may not be well tolerated in infants because of its rapid rate of solute clearance and in particular in hemodynamically unstable pediatric critically ill patients. Circuits with reduced priming volume together with monitors providing an extremely accurate fluid balance are still not commercially available [10]. Post-heart surgery patients, in particular, are a peculiar and interesting model of acute water accumulation and inflammation: they receive ultrafiltration soon after cardiopulmonary bypass wean- ing in order to remove water and inflammatory mediators before the harmful effects of inflammation and fluid overload become clinically relevant [10]. At the time of dialysis initiation, survivors tend to have less fluid overload than non-survivors, especially in the setting of 258 Z. For this reason, in children now priority indication is given to the cor- rection of water overload. In fact, differently from the adult patients where dialysis dose may play a key role, an adequate water content in small children is the main independent predictor of outcome. Nevertheless, if we consider the advantages of hemofiltration with respect to hemodialysis on the clearance of medium and higher molecular weight solutes together with the increased risk of filter clotting, predilution hemofiltration might be the preferred modality in small patients. On another side, most machines if not all, are used off label when patients below 10 kg are treated. The small number of cases, together with the limited interest of industry to develop a fully integrated device specifically designed for the pediatric popula- tion. In current practice, clinical application of dialysis equipment to pediatric patients is substantially adapted to smaller patients with great concerns about outcomes and side effects of such extracorporeal therapy. In these conditions, whereas adult critically ill patients receive renal support with modern devices and very strict safety features, smaller patients cannot rely on a very accurate delivery of therapy especially as far as fluid balance is concerned. On the other hand, it is extremely difficult to treat a small infant with a dialysis monitor providing accurate blood flow rates in the range of 10–50 ml/min and hourly ultrafiltration error below 5 ml/h. The accuracy of current systems is much lower than requested and fatal errors may occur in the very small patient. In these patients, the total blood volume ranges from less than 200 ml to about 1 l meaning that total body water content varies from 1 to 5 l: in such conditions, circuits priming volumes should be reduced to a minimum level and roller pumps should be able to run at slow speed, maintaining a good level of accuracy together with the possibility of warranting lines integrity (small roller pumps running small tubes are expected to cause a quick decline in their performance) [14 ]. This second set-up might reduce blood flow resistance and turbulence after the centrifugal pump and improve reservoir drainage when a roller pump is present. The authors promoted the concept that certain therapies should be reserved to experienced teams. Several studies, however, already showed safety and feasibility of this con- nection in the pediatric setting [18] and, even if some worries on such difficult interaction have been raised (i. Furthermore, according to these authors, fewer blood transfusions are needed and overall costs per extracorporeal membrane oxygenation run are lower. Early diagnosis, prevention, conserva- tive measures, and renal replacement therapies are all part of a common approach that must be undertaken in these high risk patients. The outcomes may vary signifi- cantly depending on the underlying disease, the severity of illness, and the time of intervention. A multidisciplinary approach should be encouraged to reach the best possible care of these patients and to utilize the highest levels of competence in each single branch of the intensive care medicine. Fluid overload and mortal- ity in children receiving continuous renal replacement therapy: the prospective pediatric con- tinuous renal replacement therapy registry. Fluid overload and fluid removal in pediatric patients on extracorporeal membrane oxygenation requiring continuous renal replacement therapy. An observational study fluid balance and patient outcomes in the randomized evaluation of normal vs. Timing of continuous renal replacement therapy and mortality in critically ill children. Inotropic support and peritoneal dialysis adequacy in neonates after cardiac surgery. Early initiation of peritoneal dialysis in neonates and infants with acute kidney injury following cardiac surgery is associated with a significant decrease in mortality. Pediatric patients with multi-organ dysfunction syndrome receiving continuous renal replacement ther- apy. Comparison of solute clearance in three modes of continuous renal replacement therapy. Continuous renal replacement therapy for children ≤10 kg: a report from the prospective pediatric continuous renal replacement therapy registry. The use of continuous renal replacement therapy in series with extracorporeal membrane oxygenation. Continuous venovenous hemofiltration with or without extracorporeal membrane oxygenation in children. Continuous renal replacement therapy with an automated monitor is superior to a free-flow system during extra- corporeal life support. Management of fluid balance in continuous renal replacement therapy: technical evaluation in the pediatric setting. Enhanced fluid management with continuous venovenous hemofiltration in pediatric respi- ratory failure patients receiving extracorporeal membrane oxygenation support. Haemofiltration in newborns treated with extracorporeal membrane oxygenation: a case- comparison study. This approach develops a culture and nursing skills base where other blood purification techniques may be possible and performed safely when needed. The didactic delivery of these topics becomes a power- ful approach when supplemented with simulation activities linked to live patient care and bedside clinical support [19, 20]. Depending on global location, regional availability, past or existing hospital contracts, leading physician input, and available budget, the choice will vary widely. Many suppliers are now offering flexible contracts where the high purchase cost is removed for acquisition of machines, but built into an anticipated consumables use contract over a number of years into the future. They all offer a version of pre-assembled disposable circuitry, colour monitor screen user interface with touch or control knob navigation and roller pumps to provide blood and fluids flow [8, 26, 27]. An internal computer manages the system reliant on pressure readings, sensors and detectors from the circuit to facilitate correct software function from the priming phase and during use. This functionality is to detect errors preventing major failure likely to cause death such as air embolism [28] or fluid imbalance [24 , 25].

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The retroviral packaging genes gag order levitra soft 20 mg what medication causes erectile dysfunction, pro cheap levitra soft online erectile dysfunction treatment maryland, pol cheap levitra soft online american express erectile dysfunction medication patents, 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.

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