K. Ashton. New England College.
Strength of evidence domains for rhythm versus rate control Domains Pertaining to SOE SOE and Number of Magnitude of Outcome Studies Risk of Consistency Directness Precision Effect (Subjects) Bias (95% CI) Rate vs buy 10mg levitra with amex erectile dysfunction after 70. Rhythm Control Using AADs Maintenance of 7 (1 buy on line levitra impotence nasal spray,473) RCT/Low Consistent Direct Precise SOE=High Sinus Rhythm OR 0. Since 6 of the 8 studies had ORs that crossed 1 (including 95% of the patients), and given significant heterogeneity, we assessed these studies as demonstrating no difference between rate- and rhythm-control strategies. CV Mortality 5 (2,405) RCT/Low Inconsistent Direct Precise SOE=Moderate OR 0. Strength of evidence domains for rhythm versus rate control (continued) Domains Pertaining to SOE SOE and Number of Magnitude of Outcome Studies Risk of Consistency Directness Precision Effect (Subjects) Bias (95% CI) CV 3 (439) RCT/Low Consistent Direct Precise SOE=High hospitalizations OR 0. Rhythm Control Using PVI Maintenance of 2 (122) RCT/Moder Consistent Direct Imprecise SOE=Low Sinus Rhythm ate Significantly better in rhythm-control strategies (OR not reported) Quality of Life 2 (122) RCT/Moder Inconsistent Direct Imprecise SOE=Insufficient ate Abbreviations: AAD(s)=antiarrhythmic drug(s); CI=confidence interval; CV=cardiovascular; OR=odds ratio; NA=not applicable; PVI=pulmonary vein isolation; RCT=randomized controlled trial; SOE=strength of evidence 108 Discussion Key Findings and Strength of Evidence In this comparative effectiveness review (CER), we reviewed 148 studies represented by 182 publications and involving 25,524 patients that directly compared rate- and rhythm-control strategies in patients with atrial fibrillation (AF). Rate-Control Drugs Our review of rate-control drugs explored the comparative effectiveness of beta blockers, calcium channel blockers, digoxin, and other antiarrhythmics in controlling ventricular rate. The 14 included studies varied in terms of the drugs involved, and the lack of multiple studies exploring similar comparisons decreased our ability to quantitatively synthesize their findings. Our findings highlight the lack of definitive data on the superiority of one beta blocker over another or against calcium channel blockers. Our findings underscore the importance of conducting studies comparing the effectiveness, tolerability and safety of different beta blockers and calcium channel blockers and in different patient populations. Table 23 summarizes the strength of evidence for the most commonly used classes of therapies and evaluated outcomes. Details about the specific components of these ratings (risk of bias, consistency, directness, and precision) are available in the Results chapter. For ventricular rate control, most comparisons were evaluated in one small study, resulting in insufficient evidence to support conclusions about comparative effectiveness. Exceptions were as follows: There was low strength of evidence that amiodarone was comparable to the calcium channel blocker diltiazem, and that amiodarone controlled ventricular rate better than digoxin, and there was high strength of evidence for a consistent benefit of verapamil or diltiazem compared with digoxin for rate control. There was insufficient evidence regarding the effect of rate-control therapies on quality of life. Summary of strength of evidence and effect estimate for KQ 1 Treatment Comparison Ventricular Rate Control Quality of Life Beta Blockers vs. Digoxin SOE=Insufficient (1 study, 47 SOE=Insufficient (No studies) patients) Beta Blockers vs. Calcium Channel SOE=Insufficient (1 study, 40 SOE=Insufficient (No studies) Blockers patients) Beta Blockers vs. Calcium Channel SOE=Insufficient (1 study, 29 SOE=Insufficient (1 study, 29 Blockers in Patients Taking Digoxin patients) patients) Sotalol vs. Metoprolol in Patients SOE=Insufficient (1 study, 23 SOE=Insufficient (No studies) Taking Digoxin patients) Amiodarone vs. Calcium Channel SOE=Low (3 studies, 271 patients) SOE=Insufficient (No studies) Blockers Amiodarone is comparable to the calcium channel blocker diltiazem for rate control Amiodarone vs. Digoxin SOE=Low (3 studies, 390 patients) SOE=Insufficient (No studies) Amiodarone controlled ventricular rate better than digoxin across 2 studies (both p=0. Summary of strength of evidence and effect estimate for KQ 1 (continued) Treatment Comparison Ventricular Rate Control Quality of Life Calcium Channel Blockers Plus SOE=Insufficient (1 study, 52 SOE=Insufficient (No studies) Digoxin vs. Digoxin Alone patients) Calcium Channel Blockers vs. SOE=High (4 studies, 422 patients) SOE=Insufficient (No studies) Digoxin Consistent benefit of verapamil or diltiazem compared with digoxin (p<0. Strict Versus Lenient Rate-Control Strategies Our review identified only one RCT and two observational studies representing secondary analyses of RCTs exploring the comparative safety and effectiveness of strict versus lenient rate- control strategies. Table 24 summarizes the strength of evidence for strict versus lenient rate control and the outcomes of interest. Details about the specific components of these ratings (risk of bias, consistency, directness, and precision) are available in the Results chapter. Across outcomes, data were limited by the number of studies and the imprecision of their findings. We based our findings on the evidence from the one RCT and then evaluated whether the observational studies were consistent or not with these findings. In general, the included studies were consistent in showing no significant difference between strict and lenient rate control with respect to mortality, cardiovascular hospitalizations, heart failure symptoms, quality of life, thromboembolic events, bleeding events, and composite outcomes. However, the RCT differed from the observational studies in showing a statistically significantly lower stroke rate with lenient rate control. Summary of strength of evidence and effect estimate for KQ 2 Outcome Strength of Evidence and Effect Estimate All-Cause Mortality SOE=Insufficient (1 study, 614 patients) CV Mortality SOE=Insufficient (2 studies, 828 patients) CV Hospitalizations SOE=Insufficient (2 studies, 1,705 patients) Heart Failure Symptoms SOE=Insufficient (2 studies, 828 patients) Quality of Life SOE=Insufficient (2 studies, 828 patients) Thromboembolic Events SOE=Low (2 studies, 828 patients) HR 0. Rate-Control Procedures Versus Drugs or Versus Other Procedures in Patients Failing Initial Pharmacotherapy Our review identified six RCTs evaluating the comparative effectiveness of a procedural intervention versus a primarily pharmacological intervention for rate control of AF, or comparing two primarily procedural interventions. We also included data from a separately published subgroup analysis of one of the RCTs. In line with our a priori definition of rate-control procedures, all studies included at least one treatment arm with radiofrequency ablation of either the AVN or His bundle, most often in conjunction with pacemaker placement. The comparison arms included a pharmacological intervention whose main purpose was to control ventricular heart rate rather than converting the underlying rhythm of AF, based on the description of outcomes; this was combined with a procedure in some studies. Tables 25 and 26 summarize the strength of evidence for rate-control procedures versus drugs and for one rate-control procedure versus another, respectively. Details about the specific components of these ratings (risk of bias, consistency, directness, and precision) are available in the Results chapter. Across outcomes and comparisons, although the included evidence was from RCTs with an overall low risk of bias and the outcomes were direct, the findings were often imprecise and based on only one or two studies. Summary of strength of evidence and effect estimate for KQ 3—rate-control procedures versus drugs Outcome Strength of Evidence and Effect Estimate Ventricular Rate Control SOE=Moderate (3 studies, 175 patients) Using different metrics, all 3 studies found that patients in the procedure arm had a significantly lower heart rate at 12 months than those on drugs All-Cause Mortality SOE=Low (2 studies, 201 patients) No significant difference CV Mortality SOE=Low (1 study, 102 patients) No significant difference Exercise Capacity SOE=Low (2 studies, 135 patients) Studies did not show significant differences between procedure and drug arms Quality of Life SOE=Insufficient (2 studies,135 patients) Abbreviations: CV=cardiovascular; KQ=Key Question; SOE=strength of evidence Table 26. Summary of strength of evidence and effect estimate for KQ 3—one rate-control procedure versus another Outcome Strength of Evidence and Effect Estimate Ventricular Rate Control SOE=Low (1 study, 40 patients) No difference between those assigned to anterior vs. Antiarrhythmic Drugs and Electrical Cardioversion for Conversion to Sinus Rhythm Our review identified 42 studies exploring the use of antiarrhythmic drugs and electrical cardioversion for conversion to sinus rhythm. Table 27 summarizes the strength of evidence for the available comparisons and evaluated outcomes. Details about the specific components of 111 these ratings (risk of bias, consistency, directness, and precision) are available in the Results chapter. Across outcomes and comparisons, although the included evidence was from RCTs with an overall low risk of bias and the evidence was based on direct outcomes, some findings were limited in terms of precision and consistency, as well as by the available number of studies. Summary of strength of evidence and effect estimate for KQ 4 Restoration of Sinus Maintenance of Sinus Recurrence of AF Treatment Comparison Rhythm Rhythm Various Methods for SOE=High (4 studies, 411 SOE=Insufficient (1 study, SOE=Low (1 study, 216 External Electrical patients) 83 patients) patients) Cardioversion (Biphasic OR 4. No No significant benefit for Significant benefit for Significant benefit of Drug Enhancement) patients given ibutilide or patients given verapamil verapamil pretreatment metoprolol pretreatment or metoprolol (p=0. Rhythm-Control Procedures and Drugs for Maintenance of Sinus Rhythm Our review identified 65 RCTs evaluating procedures for rhythm control and 18 studies evaluating the safety or effectiveness of pharmacological agents with or without external electrical cardioversion for maintaining sinus rhythm in patients with AF. Tables 28 and 29 summarize the strength of evidence for the evaluated therapies and outcomes. Details about the specific components of these ratings (risk of bias, consistency, directness, and precision) are available in the Results chapter. Across outcomes and comparisons, although the included evidence was from RCTs with an overall low risk of bias and used direct evidence, the findings were often inconsistent or imprecise, limiting our findings.
At one extrem e generic levitra 20 mg free shipping erectile dysfunction treatment austin tx, five of 11 children suffered graft loss because of recurrent disease buy levitra american express impotence after prostatectomy. H owever, m ost series have reported substantially lower recurrence rates: no recurrences in 16 adults and children, one of 34 grafts in 28 children, and two probable recurrences of 24 grafts in 20 children [4,45,46]. Graft loss occurs in 10% to 50% of patients with recurrence. HUS has been diagnosed 1 day to 15 months after transplantation (usually in less than 2 months), and the incidence of recurrence is increased in patients receiving grafts less than 3 months after their initial disease. Treatment of recurrent disease is plasma exchange for plasma or cryosupernatant, or plasma infusions, and dose reduction of cyclo- sporine. Recurrence may be prevented by aspirin and dipyridamole. FIGURE 17-36 DIFFERENTIAL DIAGNOSIS OF RECURRENT Blood film abnorm alities, m icroangiopathic hem olytic anem ia, HEM OLYTIC UREM IC SYNDROM E throm bocytopenia, and acute renal failure occur in accelerated hypertension and acute vascular rejection. A renal biopsy usually distinguishes acute vascular rejection, and malignant hypertension Thrombotic microangiopathy associated with cyclosporine should be obvious clinically. The m icroangiopathy of cyclosporine Acute vascular rejection can be difficult to differentiate from hemolytic uremic syndrome; however, glom erular pathology usually is less m arked and vascular Accelerated phase hypertension changes m ore obvious with cyclosporine toxicity. D e novo Tacrolimus- (FK-506) associated thrombotic microangiopathy hem olytic urem ic syndrom e also has been reported in patients treated with tacrolim us (FK-506). Very few patients with system ic sclerosis have received inhibitors after transplantation is unknown. Two of four patients transplantation, and the incidence of acute renal failure caused by with im m unotactoid glom erulopathy developed recurrent disease systemic sclerosis has declined with the widespread use of angiotensin- heralded by m assive proteinuria. About 20% of patients with a rarely leads to graft-related problem s; however, patients die from m alignant course of scleroderm a receiving a transplantation develop system ic com plications of ceram ide deposition. For patients with prim ary hyperoxaluria, Disease Treatment of recurrence m easures to prevent further deposition of oxalate have proved successful in controlling Focal segmental glomerulosclerosis Plasma exchange, immunoadsorption, steroids, recurrent renal oxalosis. In diabetes angiotensin-converting enzyme inhibitors, m ellitus, the pathophysiology of recurrent nonsteroidal anti-inflammatory drugs nephropathy undoubtedly reflects the sam e Immunoglobulin A nephropathy W ith crescents: plasma exchange, cytotoxics insults as those causing the initial renal failure, Henoch-Schonlein purpura? Steroids and good evidence exists that glycemic control Mesangiocapillary glomerulonephritis type I Aspirin, dipyridamole can slow the development of end-organ Mesangiocapillary glomerulonephritis type II? Plasm a exchange and im m uno- Membranous nephropathy? Cytotoxics and steroids adsorption are prom ising therapies for Anti–glomerular basement membrane disease Plasma exchange, cyclophosphamide patients with nephrosis who have recurrent Hemolytic uremic syndrome Plasma exchange, plasma infusion focal segmental glomerulosclerosis; however, Antineutrophil cytoplasm antibody–associated vasculitis Cyclophosphamide and steroids these therapies do not provide sustained Diabetes Glycemic control remission [6,7]. In all these cases, establishing Oxalosis Aggressive perioperative dialysis, hydration, low oxalate a diagnosis of recurrent disease is critical in diet, low ascorbic acid diet, phosphate supplements, identifying a possible treatm ent m odality. Even excluding these conditions, the overall rate of recurrence of glomerulonephritis is substantially increased in living related donors, Focal segmental glomerulosclerosis with risk factors for early recurrence and patients should be m ade aware of this risk. For fam ilial Henoch-Schonlein purpura diseases, the risk of recurrence is even higher (eg, som e fam ilies with im m unoglobulin A disease and hem olytic urem ic syndrom e). Mesangiocapillary glomerulonephritis type I Finally, recurrent glom erulonephritis has been reported in up to Mesangiocapillary glomerulonephritis type II with risk factors (familial immunoglobulin A nephropathy and hemolytic uremic syndrome) 30% of renal isografts, with disease onset between 2 weeks and 16 years after grafting. Tejani A, Stablein DH: Recurrence of focal segmental glomerulonephritis 7. Transplantation plantation in prim ary hyperoxaluria type 1. Experiments in the form of randomized controlled trials provide the most per- suasive evidence for action in public health. By 2010, 18 such trials in Africa, Asia and Europe had shown that the participation of outreach workers, lay health workers, com- munity midwives, community and village health workers, and trained birth attend- ants collectively reduced neonatal deaths by an average of 24%, stillbirths by 16% and perinatal mortality by 20%. Community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Cochrane Database of Systematic Reviews (Online), 2010,11:CD007754. PMID:21069697 iv trials clearly do not give all the answers – for instance, the benefts of these interven- tions in reducing maternal mortality, as distinct from morbidity, are still unclear – but they are a powerful argument for involving community health workers in the care of mothers and newborn children. Tese rigorous investigations have the potential to beneft millions around the world. Tey confront the challenge presented by just one of the MDGs, but they cap- ture the general spirit of this report – to promote investigations in which creativity is harnessed by the highest-quality science in order to deliver afordable, quality health services and better health for everyone. More than that, the process of discovery is a source of inspiration and motivation, stirring ambitions to defeat the biggest problems in public health. Tis is the purpose of Research for universal health coverage. Tis report is for everyone concerned with understanding how to reach the goal of universal health coverage – those who fund the necessary research, those who do research and who would like to do research, and those who use the evidence from research. It shows how research for health in general underpins research for universal health coverage in particular. Understanding how to make progress towards achieving the MDGs is central to this report. As the 2015 deadline draws closer, we are looking for ways to improve all aspects of health, working within and beyond the MDG framework. And we are investigating how better health can contribute to the larger goal of human development. In this broad context, I invite you to read Research for universal health coverage. Dr Margaret Chan Director-General World Health Organization v Contents Message from the Director-General iv Executive summary xi 1. The role of research for universal health coverage 5 Developing the concept of universal health coverage 6 Investigating fnancial risk protection 11 Investigating the coverage of health services 15 Equity and universal health coverage 19 Coverage of health services: quality as well as quantity 20 Conclusions: research needed for universal health coverage 21 2. The growth of research for universal health coverage 31 Creativity everywhere 35 Research ascending 35 Growing unevenly 42 The value of health research 46 Conclusions: building on the foundations 47 3. How research contributes to universal health coverage 57 Case-study 1 61 Insecticide-treated mosquito nets to reduce childhood mortality Case-study 2 63 Antiretroviral therapy to prevent sexual transmission of HIV Case-study 3 65 Zinc supplements to reduce pneumonia and diarrhoea in young children vii Case-study 4 67 Telemedicine to improve the quality of paediatric care Case-study 5 69 New diagnostics for tuberculosis Case-study 6 71 The “polypill” to reduce deaths from cardiovascular disease Case-study 7 73 Combination treatment with sodium stibogluconate (SSG) and paromomycin compared to SSG monotherapy for visceral leishmaniasis Case-study 8 75 Task shifting in the scale-up of interventions to improve child survival Case-study 9 77 Improving access to emergency obstetric care Case-study 10 79 Conditional cash transfers to improve the use of health services and health outcomes Case-study 11 81 Insurance in the provision of accessible and afordable health services Case-study 12 82 Afordable health care in ageing populations Conclusions: general lessons drawn from specifc examples 84 4. Building research systems for universal health coverage 95 Setting research priorities 96 Strengthening research capacity 98 A framework for strengthening capacity 99 Creating and retaining a skilled research workforce 103 Ensuring transparency and accountability in research funding 105 Building research institutions and networks 107 Defning and implementing norms and standards 110 Ethics and ethical review 110 Reporting and sharing research data, tools and materials 110 Registering clinical trials 110 Using evidence to develop policy, practice and products 113 viiiviii Translating evidence into policy and practice 113 Monitoring and coordinating research, nationally and internationally 116 Financing research for universal health coverage 117 National and international governance of health research 118 Conclusions: building efective research systems 118 5. Research has the power to address a wide range of questions about how we can reach universal coverage, providing answers to improve human health, well-being and development. The creativity and skills of researchers should be used to strengthen investigations not only in academic centres but also in public health programmes, close to the supply of and demand for health services. To make the best use of limited resources, systems are needed to develop national research agendas, to raise funds, to strengthen research capacity, and to make appropriate and effective use of research findings. In 2005, all WHO Member States made the commitment to achieve universal health coverage. Te commitment was a collective expression of the belief that all people should have access to the health services they need without risk of fnancial ruin or impoverishment. Working towards universal health coverage is a powerful mechanism for achieving better health and well-being, and for pro- moting human development. Chapter 1 explains how the resolution adopted by all WHO Member States embraces the two facets of universal health coverage: the provision of, and access to, high-quality health services; and fnancial risk protection for people who need to use these services. Te term includes ways of taking action on social and environmental determinants both within and beyond the health sector.
Replace the remaining deficit over the next 24–36 hrs Perform serial neurologic examinations (prescribed rate of correction can be decreased as symptoms improve) Measure serum and urine electrolytes every 1–2 hrs *If UNa + U K is less than the concentration of PNa generic 10 mg levitra impotence underwear, then water loss is ongoing and needs to be replaced generic levitra 10mg without a prescription erectile dysfunction exercises wiki. Jacobson H R: Functional segm entation of the m am m alian nephron. Berl T, Schrier RW : Disorders of water m etabolism. Berl T, Anderson RJ, M cDonald KM , Schreir RW : Clinical Disorders Publishing Co. Kokko J, Rector F: Countercurrent m ultiplication system without 18. Gullans SR, Verbalis JG: Control of brain volum e during hyperosm o- active transport in inner m edulla. Knepper M A, Roch-Ram el F: Pathways of urea transport in the m am - 19. Zarinetchi F, Berl T: Evaluation and m anagem ent of severe hypona- trem ia. Lauriat SM , Berl T: The H yponatrem ic Patient: Practical focus on 5. Zim m erm an E, Robertson AG: H ypothalam ic neurons secreting vaso- 21. Ayus JC, W heeler JM , Arieff AI: Postoperative hyponatrem ic pressin and neurophysin. Bichet DG: N ephrogenic and central diabetes insipidus. Laureno R, Karp BI: M yelinolysis after correction of hyponatrem ia. Bichet DG : Vasopressin receptors in health and disease. Kum ar S, Berl T: Disorders of serum sodium concentration. Dunn FL, Brennan TJ, N elson AE, Robertson GL: The role of blood 24. In Fluid & Electrolytes, osm olality and volum e in regulating vasopressin secretion in the rat. N ew York: m utations in the vasopressin-neurophysin II gene in 17 kindreds with M cGraw H ill, 1994. In Fluid & Electrolytes, Physiology and Pathophysiology. Edited by Jacobson H R, Striker GE, Appleton & Lange, 1991:98. H alterm an R, Berl T: Therapy of dysnatrem ic disorders. Barrett T, Bundey S: W olfram (DIDM O AD) syndrom e. H oltzm an EJ, Ausiello DA: N ephrogenic Diabetes insipidus: Causes 13. Veis JH , Berl T, H yponatrem ia: In The Principles and Practice of revealed. Bichet D, O ksche A, Rosenthal W : Congential N ephrogenic Diabetes St. Berl T, Schrier RW : Disorders of water m etabolism. Philadelphia: Lippincott-Raven, nephrogenic diabetes insipidus hom ozygous for m utations in the 1997:52. Verbalis JG: The syndrom e of ianappropriate diuretic horm one secre- 31. Boston: Little, Brown, and nephrogenic diabetes insipidus with partial vasopressin response. McCarthy Rajiv Kumar agnesium is an essential intracellular cation. Nearly 99% of the total body magnesium is located in bone or the intracellular M space. M agnesium is a critical cation and cofactor in numerous intracellular processes. It is a cofactor for adenosine triphosphate; an important membrane stabilizing agent; required for the structural integrity of numerous intracellular proteins and nucleic acids; a substrate or cofac- tor for important enzymes such as adenosine triphosphatase, guanosine triphosphatase, phospholipase C, adenylate cyclase, and guanylate cyclase; a required cofactor for the activity of over 300 other enzymes; a regulator of ion channels; an important intracellular signaling molecule; and a modulator of oxidative phosphorylation. Finally, magnesium is intimately involved in nerve conduction, muscle contraction, potassium transport, and calcium channels. Because turnover of magnesium in bone is so low, the short-term body requirements are met by a balance of gastrointestinal absorption and renal excretion. Therefore, the kidney occupies a central role in magnesium balance. Factors that modulate and affect renal magnesium excretion can have profound effects on magne- sium balance. In turn, magnesium balance affects numerous intracellular and systemic processes [1–12]. In the presence of normal renal function, magnesium retention and hypermagnesemia are relatively uncommon. Hypermagnesemia inhibits magnesium reabsorption in both the proximal tubule and the loop of Henle. This inhibition of reabsorption leads to an increase in magnesium excretion and prevents the development of dangerous levels of serum magnesium, even in the presence of above-normal intake. However, in familial hypocalciuric hypercalcemia, there appears to be an abnormali- C H A P T ER ty of the thick ascending limb of the loop of Henle that prevents excre- tion of calcium. In familial hypocalciuric hypercalcemia, mild hypermagnesemia does not increase the renal excretion of magnesium. A similar abnormality may be caused by lithium [1,2,6,10]. The renal excretion of magnesium also is below normal in states of hypomagnesemia, decreased dietary magnesium, dehydration and volume depletion, hypocalcemia, hypothyroidism, and hyperparathyroidism [1,2,6,10]. Total body M g content is about 24 g (1 m ol) per 70 kg. M g in Bone 53 530 12720 bone is adsorbed to the surface of hydroxy- Muscle 27 270 6480 apatite crystals, and only about one third is Soft tissue 19. O nly 1% to 3% of the total intracellular M g exists as the free ionized form of M g, which has a closely regulated concentration of 0. Proteins, enzymes, Total cellular M g concentration can vary from 5 to 20 m m ol, citrate, Endoplasmic depending on the type of tissue studied, with the highest M g con- ATP, ADP reticulum centrations being found in skeletal and cardiac m uscle cells. O ur – understanding of the concentration and distribution of intracellular M embrane – M g has been facilitated by the developm ent of electron m icroprobe proteins – analysis techniques and fluorescent dyes using m icrofluorescence spectrom etry.
For example buy levitra 20 mg line erectile dysfunction 16 years old, an understand- ing of the mechanisms by which repeated exposure to drugs of abuse increases their stimulant and rewarding properties HERPES SIMPLEX VIRUS: THE PROTOTYPIC in animal models will almost certainly lead to new ways of VECTOR treating addiction in humans order line levitra erectile dysfunction treatment atlanta ga. If we are able to decipher the molecular events underlying long-term changes in neuro- HSV possesses multiple features that make it an ideal vector transmitter release, we will find new approaches to diseases for delivery of genes into the nervous system. In particular, such as the epilepsies, in which neurotransmitter release is it accepts large molecules of exogenous DNA; it infects non- altered. Knowledge of the molecular means by which neuro- dividing cells from a wide range of hosts with high effi- transmitters shape neuronal development and plasticity, or ciency; it enables strong expression of foreign genes; it is how trophic factors regulate neuronal health, will lead to episomal, and thereby does not cause integration effects; insights into how defects in these pathways cause specific its infection of postmitotic cells is persistent; and HSV-1 psychiatric and neurodegenerative diseases. Be- Unfortunately, the brain does not yield easily to genetic cause of these characteristics of HSV-1, and because it is intervention. The terminally differentiated state of most neurotropic, it is currently one of the best viral vectors avail- neurons in the brain precludes the use of vectors, such as able for functional analysis of genes in the nervous system. In addition, the molecular mechanisms of specific brain disorders may be Amplicon vs. Genomic HSV-1 Vectors restricted to subsets of neurons at specific times during de- There are two types of replication-deficient HSV vectors: velopment and maturity. Therefore, strategies for manipu- those in which the foreign DNA of interest is cloned into lating gene expression in the brain must utilize vectors that the viral genome itself (genomic vectors), and those that are persist stably in postmitotic cells and that can be targeted composed of a plasmid carrying minimal HSV sequences both spatially and temporally in the nervous system. A num- that allow it to be packaged into virus particles with the aid ber of such gene delivery systems have been developed over of a helper virus (amplicon vectors). Rather than giving a superficial overview within the wild-type HSV genome are dispensable for its of the field, this chapter highlights the use of herpes simplex growth in cells in vitro. This type of geneti- compares and contrasts HSV-mediated genetic intervention cally engineered genomic vector has been used by a number of investigators and is described in detail by Fink et al. The idea of the amplicon vector originated with the dis- covery of defective HSV-1 particles (3,4) that appeared in Rachel L. Examination of the 254 Neuropsychopharmacology: The Fifth Generation of Progress genomes of these defective HSV-1 particles revealed that TABLE 20. CHRONOLOGY OF IMPROVEMENTS IN they carried only a minimal subset of DNA sequences from AMPLICON VECTOR PACKAGING SYSTEM the wild type genome (3–5). The plasmid sequences of methods to purify and concentrate the virus result in that were packaged into virus particles consisted primarily amplicon/helper ratios of up to 100:1 (previously 1:100) and titers of up to 2 × 108 infectious units/mL of 150-kilobase (kb) concatamers of the original plasmid 1997—Helper-free packaging by transient transfection of HSV (3–6). One disadvan- tage is that production of amplicon vectors requires a co- propagated HSV helper virus, resulting in viral stocks that dure (Table 20. The most widely used second-generation cytotoxic effects of these stocks limited the amount of vector helper virus was HSV-1 tsK, with a temperature-sensitive that could be used to infect cells. Even though the replica- single-base mutation in the ICP4 (IE3) gene. Because re- tion-incompetent helper viruses could not cause lytic infec- vertants of this mutant arose at a finite frequency during tions in normal cells, cytopathic effects resulted both from the packaging procedure, lytic virus was present in some proteins present in the HSV-1 particles and from expression preparations (7,8). The frequency of revertants was de- of HSV-1 immediate-early (IE) genes (7). Occasionally, creased with the development of an efficient packaging sys- wild-type HSV-1 revertants appeared during the amplicon tem using a deletion mutant of IE3 (8) as helper virus. However, recent improvements in the pear, albeit at a greatly reduced frequency, presumably as amplicon packaging procedure, which are discussed in the a result of recombination between the helper virus and the following section, have largely overcome these problems. Historically, IE3 mutants Present-Day Amplicon Vectors: have been preferred because they express fewer HSV-1 genes Advantages and Disadvantages under nonpermissive conditions than do ICP27 mutants. Genomic and plasmid defective HSV-1 vectors have been However, Lim et al. These studies have been promising, but they have with no increase in cytotoxicity. In addition, wild-type lytic also revealed limitations of the current HSV vector systems. D30EBA, sharing little sequence with the IE2-containing Moreover, as noted above, nonspecific cytopathic effects of fragment present in the permissive host. Finally, lack of persis- helper virus, the stocks derived from transfection of the tence of high expression levels from the viral recombinants packaging cells followed by superinfection with helper virus has hampered long-term in vivo studies and has limited the are passaged three times on the permissive host. The recom- usefulness of the vectors for both experimentation and gene binant vector is packaged as long concatamers, which con- therapy. Therefore, the efficiency of the initial transfection of vector DNA into the packaging line is critical to the success of the packaging. Since then, we have achieved vector/helper ratios greater than 100. For a viral vector to have utility for gene therapy, cyto- pathic effects of the virus must not outweigh the beneficial effects of the transgene. Unfortunately, in the past, investi- gators have had difficulty generating nontoxic HSV-based replication-defective vectors. Significant necrosis, often accompanied by inflammation and gliosis, was identified at injection sites with some genetically engineered HSV-1 vectors used for in vivo studies (see, e. However, the achievement of a more favorable ratio of vector to helper, and the virtual elimination of wild-type virus in the vector preparations (10) has greatly reduced the cytotoxicity of present-day de- fective HSV-1 amplicon vectors (see, e. An additional improvement to the packaging procedure, the banding of the virus on a sucrose step gradient, followed by a high-speed centrifugation to pellet the virus, has reduced FIGURE 20. Different vector constructions for coexpressing further the cytotoxicity of the virus preparations. Glutamate receptor GluR1 and green fluorescent protein (GFP)are used as examples. A troubling problem that has not yet been resolved for any viral vector used in the brain, except perhaps for the An improvement in gene transfer methods in general has lentiviral vector (see below), is that of persistence of expres- been the incorporation of the gene for the green fluorescent sion. Numerous investigators have had the experiences re- protein (GFP) into many vectors. In the example shown, the objec- estingly, superinfection with helper virus 5dl1. Apparently, GFP) by putting an internal ribosome entry site (IRES) transactivating factors provided by the helper virus reacti- between the two genes, which are then transcribed from the vated transcription of the transgene. The IRES enables independent translation Two recent developments suggest that the problem of of the two coding sequences even though they are present persistence of expression is not insoluble. In theory, the two fragment of the tyrosine hydroxylase promoter to drive re- coding sequences should be expressed at similar levels, but porter gene expression in an HSV-1 amplicon vector re- in practice the translation of one of the two coding se- sulted in prolonged gene expression in vivo (16), suggesting quences on the mRNA often occurs at the expense of the that neuronal, unlike viral, promoters in HSV-1 vectors other. Alternatively, they can be expressed from two inde- have the potential to produce stable gene expression. Addi- pendent transcriptional units, in a bicistronic vector. The tionally, the development of hybrid amplicons that incorpo- addition of an extra transcriptional cassette to the vector rate elements that allow autonomous replication of the epi- makes it larger and more unwieldy to use; and the level of some (17) or that incorporate adeno-associated virus (AAV) transcription from one promoter is independent of the level elements for genomic integration of the amplicon (18–20) of transcription from the other, so that the transgenes may have resulted in vectors that support long-term gene expres- be expressed at very different levels. Third, the GFP can be sion both in vitro and in vivo. This is a trickier construction, since the can be concentrated to very high titers ( 1010/mL). How- two coding sequences must be placed in frame with each ever, the use of adenovirus vectors continues to be restricted other.