By R. Sebastian. University of Nebraska, Lincoln.

Oligonucleotides act by altering expression of endogenous genes in cells by a variety of mechanisms (see Figure 4 buy caverta pills in toronto. There are three important factors regarding nonviral vectors that can be ampli- fied in prokaryotic or eukaryotic cells for gene therapy: (1) the size of the insert accepted best caverta 100mg, (2) how to get the genetic material into cells efficiently buy 50mg caverta fast delivery, and (3) how to maintain the genetic material inside the cell in order to achieve long-term expression. Nonviral vectors are any type of vector that does not involve a viral particle that can alter gene expression in a cell. They generally contain a promoter and coding sequence that results in expression of a therapeutic protein. Oligonucleotides, or more stable analogs such as phosphorothioates, contain 10 to 25 bases. Size of Insert The size of the insert accepted varies considerably among the different nonviral vectors that replicate in cells. Bacteria can amplify plasmids, bacteriophage, cosmids, or bacterial artificial chromosomes. More recently, the production of a human artificial minichromosome was reported, although its transfer into cells was very inefficient. For ex vivo transfer, genes are usually transferred into the cell by using calcium phosphate co-precipitation, electroporation, cationic lipids, or liposomes. Efficient in vivo transfer is somewhat more difficult to achieve than ex vivo gene transfer. Selective delivery (targeting) of a nonviral vector to a specific organ or cell type would be desirable for some applications. Stabilization of Nonviral Vectors in Cells A major problem with nonviral vectors is transient gene expression, since the genetic material transferred into the cell is unstable. Some investigators have placed origins of replication derived from viruses into nonviral vectors. Plasmids must be engineered to express any proteins necessary to activate the origin of repli- cation. Plasmids containing these replication origins and relevent appropriate proteins activating origins are main- tained longer in cells in vitro and in vivo than plasmids that do not contain these sequences. Artificial chromosomes have elements that stabilize genetic material in a cell and should not have problems of instability. If difficulties in amplifying and transferring artificial chromosomes into cells can be overcome, such vectors should be maintained stably in a cell. There is little quantitative data regarding the efficacy of expression from different promot- ers in vivo. Gene therapy with plasmid vectors has been used to attempt to treat cystic fibrosis (see Chapter 3) and cancer in humans (see Chapter 10). A major advantage of using nonviral vectors is the lack of risk of generating a wild-type virus via recombination. In addi- tion, 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.

This effect was considered will allow the patient time to become accustomed to minor and all birds remained alert buy 50 mg caverta otc. Return to normal the collar buy cheap caverta on line, and it will not damage the wings while the motor coordination occurred within two to four hours patient is struggling to escape the device purchase caverta 50mg. No gastrointestinal effects were day, the collar should be reversed such that the cone observed with this agent. Food and water should be placed on a pedestal to ten times this dose are safe in birds. The patient’s weight should be closely monitored to assure that an adequate amount of food Buprenorphine hydrochloridev is another opioid with and water is being consumed. The collar should be agonist/antagonist activity that appears to be effec- tive in controlling pain in avian patients. Vet Surg 18:269- Tissue reaction to five suture materi- Assoc 189:1017-1019, 1986. Board certified surgeons are becoming attracted to the field for the purpose of developing 41 and refining avian procedures. Procedures that were once considered impossible are now performed on a routine basis. The most substantial limitation to soft tissue surgery of the abdomen is the small size (<100 grams) of many avian patients. Surgery of the thoracic area, even in large companion birds, pre- sents a similar problem, in that the organs of interest are covered by the sternum and heavy musculature. Continued improvements in the endoscopic surgical equipment available in human medicine will un- doubtedly improve the surgeon’s ability to perform R. Harrison The avian surgeon should practice surgical tech- niques on cadavers prior to performing the proce- dures on clinical patients. The delicate avian tissues tear in the presence of slight autolysis; therefore, the use of fresh specimens will give the surgeon an ap- preciation of avian tissue characteristics and allow the surgeon to explore the capabilities of surgical instrumentation. When necropsies are necessary, the clinician should approach this procedure from the perspective of a surgeon rather than of a pathologist, by dissecting and reviewing anatomy from a regional approach rather than by performing the necropsy strictly from the traditional ventrodorsal approach. Surgery of the Skin In small birds (eg, Passeriformes), constricting fibers may be visualized using the operating microscope (see Figure 43. A bent 25 ga needle is helpful for The skin and subcutaneous tissues of birds differ removing constricting fibers. Birds have relatively thin, elevate the fiber, which can then be cut by gently dry epidermis, and the dermis is attached to the rolling the needle such that the beveled edge severs underlying muscle fascia with little subcutaneous the fiber. Compared to mammals, the skin exposed tendons may heal without incident once the is only loosely attached to underlying structures, fibers are removed. A hydroactive dressing should be except in the distal extremities where it is firmly placed on any wounds created by the fibers to prevent adherent to underlying bone. Neonates (especially macaws and Eclectus Parrots) Passerine Leg Scales may develop constrictive toe lesions that can result in avascular necrosis of the digit (see Color 30). Pro- Passerine leg scale syndrome is characterized by the posed etiologies for these include low humidity, egg- development of abnormally large scales of the legs 25 related strictures or ergot-like intoxication. In- and feet, possibly as a result of mite infection or creasing the environmental humidity or providing malnutrition (see Chapter 43). These scales can coa- hot moist compresses and massage may be effective lesce and act as a constricting band. More ad- dispose the bird to bacterial pododermatitis (usually vanced lesions require surgical intervention. If present, the shiny, convex cumferential indentation is treated using magnifica- carapace of the female Knemidocoptes mite can usu- tion to remove the constricting tissue (Figure 41. In most A tourniquet fashioned from a rubber band held instances, lesions resolve after treatment with iver- tightly with a mosquito hemostat may be used to mectin or correction of nutritional deficiencies. In control hemorrhage for short periods until the injury severe cases, it may be necessary to surgically de- is properly treated. Hemostatic agents including ra- bride the proliferative scales to prevent vascular diocoagulation should be avoided. A 22 or 25 ga needle with the point bent to the digits is minimal, and anything that interferes to a 90° angle can be used to lift the scales and scabs, with proper blood flow may predispose the digit to which can then be grasped with the micro-forceps. A circumferential anastomosis of the skin is then performed by placing one or two sutures in the sub- Toe Necrosis (Constricted Toe Syndrome) cutaneous tissues to provide skin apposition without Avascular necrosis of digits may occur secondary to tension. Skin sutures should be placed shallow below circumferential constriction caused by fibers, scabs the epidermis and be sufficiently tight to appose the or necrotic tissue (see Color 24). Su- cause edema and if untreated, sloughing of the digit tures placed too deeply will cause the skin edges to distal to the constriction. This condition is generally4 evert, exposing subcutaneous tissue and delaying not life-threatening, and amputation should be con- healing. When the skin edges are apposed, a two to sidered only after less aggressive therapies have three millemeter release incision should be made at failed. Removal of the offending tissue or fibers and the site of the anastomosis on both the lateral and supportive care are frequently successful. These incisions allow necrosis of the digits has been described in passerine swelling without constriction. Scabs should be debrided is applied to prevent scab formation, which could or incised to prevent vascular compromise, and hy- result in reformation of the constriction. Feather Cysts follicle, feather growth becomes asymmetrical and the feather may grow in a curled fashion inside the Feather cysts are generally the result of trauma to follicle, resulting in a feather cyst. Fulguration with a radiosurgical unit has been reported to be successful Feather cysts may occur within any feather follicle, in some cases; however, the depth of destruction is but those on the wing and tail are the most challeng- difficult to control, resulting in damage to adjacent ing to the surgeon. These damaged follicles can then develop common in Norwich, Gloucester and their cross- feather cysts. These birds have been genetically selected to appear to improve the long-term clinical results. The feather development have been associated with entire follicle, including any bony attachments, trauma, malnutrition and viral, bacterial or parasitic should be excised. As adjacent feathers begin Xanthomatosis is characterized by the deposition of to regrow, debris should be gently removed by flushing a rubber-like proteinaceous material within the skin with warm sterile saline several times daily. Xanthomas at the With a single cyst or a large feather, the follicle may wing tip may cause the wing to droop, resulting in be saved by marsupializing the lining of the cyst with trauma to the mass. An incision is made Amazon parrot) and dietary management should be centered on the cyst, parallel to the direction of used in combination with surgical excision of the feather growth. Redun- els should be closely monitored because they are dant tissue is excised and the follicle is thoroughly usually elevated in birds with xanthomatosis and lavaged with sterile saline. The margin of the cyst is should be medically reduced to a normal level prior then sutured to the skin using a simple continuous to surgery. A monopolar, wire electrode functions well for re- Feather cysts of the tail may be severe and disfigur- moval of xanthomatous masses. The wound may be pro- dissection to the coccygeal vertebrae allows disar- tected with tissue adhesive or a hydroactive dress- ticulation at the sacrococcygeal junction without en- ing, which should be changed every three to five tering the cloaca.

An ovary and oviduct tion in maturing hens causes a hierarchy of follicles can regress to a point where it is difficult to deter- to develop order caverta 50 mg on line, giving the ovary the appearance of a mine if a hen has ever been reproductively active cheap caverta 100 mg on line. As the The oviduct consists of five microscopically distin- breeding season approaches cheap caverta 100mg otc, the follicles undergo a guishable regions: infundibulum, magnum, isthmus, period of rapid growth with the deposition of yolk uterus (shell gland) and vagina (Figure 29. At this point and ventral ligaments attach the oviduct in the peri- the yellow yolk is clearly visible through the highly 79 toneal cavity. The large follicle is sus- ment is clearly visible crossing the cranial division of pended by a stalk. The cranial oviduct and stronger smooth muscle contrac- hypertrophied granulosa cells are metabolically ac- tions in the uterus and vagina move the ovum down tive for several days and may not be reabsorbed until and the sperm up the reproductive tract. Oviduct eight to ten days post-ovulation in the chicken, and 100 transit time varies among species and is approxi- up to several months in the Mallard Duck. Similar transit times post-ovulatory follicle is thought to secrete non- are discussed in companion and aviary birds, with steroidal hormones that are involved in oviposition 86 the egg spending varying but proportional times in and nesting behavior. During the non-breeding season, the ovarian follicles The cranial infundibulum consists of a thin, nearly normally collapse and exhibit atresia. Two kinds of transparent finger-like funnel that engulfs the ovum atresia have been described. More curs when the follicle wall ruptures and yolk is harm- distally, the infundibular wall thickens as it becomes lessly released into the peritoneal cavity where it is tubular. Invasion atresia involves granulosa and seven centimeters long, while in the Brown Kiwi it theca cells invading the ovum with subsequent in extends the width of the peritoneal cavity to receive situ yolk absorption. The earliest detectable indica- 86 oocytes from functional left and right ovaries. Other species may have fundibulum, where sperm may reside in glandular only one set of these glands. Production glands remain fertile for 7 to 14 days in the chicken of the chalaziferous layer of the albumen and the and for 40 to 50 days in the turkey. It is the largest and most coiled portion of Primary oogenesis begins in the embryo when secon- the oviduct and deposits most of the albumen, so- dary oocytes are formed. Meiosis is arrested until dium, magnesium and calcium used in egg develop- adult life when follicles become active and grow in ment. The egg may remain in the magnum for three three phases, the first of which can last months to hours. The isthmus has less well developed circu- two lasts about 60 days in the domestic hen, and lar muscle and glandular tissue compared to the during this period some yolk is deposited in vacuoles magnum. This stage corresponds with ovar- ian regression that occurs in the non-breeding season The short uterus has numerous leaf-like lamellae in free-ranging species. This part of the uterus is ovoid in shape are both produced by the adenohypophysis under the and holds the egg during shell deposition. Stage three involves cranial aspect of the uterus is difficult to differentiate rapid yolk deposition and normally occurs in free- from the isthmus. Courtship 20 to 26 hours and receives salts, water, the shell and and nest-building activity seem to precede stage shell pigment. The uterus is highly vascularized dur- three follicular development, which terminates with ing egg laying and must be carefully manipulated either normal follicular atresia or ovulation. The egg is oriented in ers of yolk, the oocyte, perivitelline lamina, granu- the uterus with its sharp end pointing caudally. Ovarian thecal most species the egg is laid in this direction, although and interstitial cells produce estrogen while the in some species the egg turns in the uterus just before granulosa cells produce progesterone. The vagina is distinguishable by its numer- causes extrusion of the first polar body, the follicular ous thin folds of mucosa. Extrusion of the some Anseriformes, the vagina is separated from the second polar body occurs in the infundibulum, and cloaca by a membrane that deteriorates at sexual the ovum is formed. These cells continue to produce progester- nal lumen in seconds during normal oviposition. During this time, the turbed during oviposition may retain an egg in the ovary and accessory reproductive tissues, such as the vagina for four days, followed by a normal egg deliv- oviduct and comb, regress. Prolactin secreted by the anterior pituitary Gallinaceous hens can store sperm in the spermatic stimulates the production of “crop milk” in both gen- fossulae (sperm host glands) at the uterovaginal ders of Columbiformes. Photorefractoriness is then terminated by lation or, if given 36 hours before expected ovulation, shorter daylight periods. The extrapolation of any data Domestic hens are continuous layers (indeterminate collected in gallinaceous birds should be applied to layers) and under optimum conditions are reproduc- companion bird species with caution. Hormonal mechanisms con- is used to prevent egg laying, it should be adminis- trolling continuous egg production have been artifi- tered when a complete clutch has been laid. In general, the seasonal hypertrophy fluenced by increasing photoperiod in temperate cli- of the oviduct in free-ranging birds is dependent on mates. Photoperi- prolactin also interact with estrogen in stimulating odic pathways are controlled by light passing the growth and secretory activity of the oviduct. In through the eye via the optic nerve to the hypothala- the oviduct, estrogens influence the synthesis of mus, and by light passing through the spongy calvar- oviductal proteins, oviduct growth and the formation ium stimulating the hypothalamus or pineal body. Androgens in estrogen-primed Photoperiod also affects the time of ovulation and birds influence the synthesis of proteins in the ovi- oviposition. In poultry, the maximum effect of photo- duct and in conjunction with estrogen initiate medul- stimulation occurs when birds are provided 12 to 14 lary ossification. Ovum transport in the oviduct is primarily accom- Because reproductive activity continues even when plished by contractions of the oviduct in response to hens are placed in continuous darkness, other factors a stretch stimulus. The effect of dwelling species, such as the budgerigar and Zebra oxytocin, also produced by the posterior pituitary, in Finch, the rostral pituitary is constantly stimulated inducing premature oviposition may be mediated in by the hypothalamus to release gonadotropins except vivo by prostaglandins. It is likely that oviposition is when inhibited by negative external conditions such a complicated process involving neurohypophyseal as drought. During these dry conditions when food hormones, prostaglandins and hormones of the pre- would be scarce, the hypothalamic secretions sup- and postovulatory follicles. Be- fore expulsion of the egg can occur, the abdominal Ovulation in the domestic hen occurs shortly after muscles and cervix must relax. High- growth, bill growth and aggressive male-type behav- fat, low-calcium diets exacerbate a calcium defi- ior (territoriality). Affected crease again in both genders if a second clutch of eggs budgerigar hens have been shown to have normal is laid. Prolactin levels increase in both genders dur- for inactivating estrogens, and it has been suggested ing egg laying, peak during incubation and then that impaired liver function may be responsible for decline to a resting level. Calcium Metabolism Other Metabolic Changes High levels of circulating calcium are needed for shell Hematogenic changes associated with egg laying in- formation. Estrogen increases total plasma calcium clude a slight increase in white blood cell count, by increasing the production of blood calcium-bind- packed cell volume, total serum solids and total pro- ing proteins. Total serum solids and total protein are in- birds, the calcium levels can become extremely high, creased because of a need for protein for calcium reaching levels of 30 mg/dl.

Interobserver agreement is high and determining the five factors is useful to establish a profile of cognitive impairment (38) 50 mg caverta mastercard. It is completed by patients to assess their perception of their own ca- pacities regarding physical efforts purchase caverta overnight delivery, self care trusted 100 mg caverta, emotional control, relation- al possibilities, and cognitive processes. The evaluation is performed by the patient, a member of the family and a care provider. A score of anosognosia is obtained by the difference between the score of the pa- tient and that established by the family member and/or care provider. The interobserver repro- ducibility is, however, poorer for items investigating cognitive dimen- sions, which are more abstract than the motor-related items (41). This is attributable to the difficulty of consid- eration of the veritable repercussions of psychological, behavioral and so- cial factors. Along with the generic evaluation tools of handicap and quality of life, specific tools have been developed for patients with head in- juries. It was cre- ated to assess the overall functional outcome of brain trauma victims, regardless of severity. It describes five levels: death, persistent vegeta- tive state, severe handicap, moderate handicap and good recovery. Both reproducibility and sensitivity are good, at least during the ini- tial months. In contrast, it makes a real contribution to epidemiological studies and within the framework of long-term follow-up, to improve, for example, the measures of public health and social politics de- signed for subjects with brain injuries. The Disability Rating Scale (44), which is also specific, has four cat- egories: level of consciousness, cognitive capacities for self care, physical dependence to others, and social reinsertion (work, house- keeping, school activity). It is a discriminating tool even though, in some cases, it does not cover all aspects of handicaps. The Rivermead Head Injury Follow Up Questionnaire (48) is based upon comparison by patients of their life since the trauma with re- gard to their life previously. Ten items are investigated including per- formance of daily living activities, ability to follow a conversation, to participate in social and leisure activities, professional activity, and relationships with spouse, family, and friends. The European Head Injury Chart (49) has been developed in vari- ous languages of the European Community. It essentially involves a standardized, comprehensive observation, containing 175 items to assess the changing phases of head injury. The time required to complete this test is long (2 to 3 hours) limiting its practical uti- lization. Consequently, there are numerous types and degrees of im- pairment and disability. Furthermore, there is substantial subjective fall- out from the fatigue and depression that are so frequent in this disease. Both the development of new therapeutic protocols and the adaptation of rehabilitation protocols throughout the course or the disease require sen- sitive and prognostic evaluation tools. Technical perfec- tioning of imaging studies results in improved clinical and prognostic cor- relations in advanced stages, notably regarding cognitive impairment. It involves attributing values to seven “functional parameters” corresponding to various potentially damaged neurological systems and to functional capacities primarily regarding am- bulation. The final score obtained by combination of these various para- meters ranges from 0. The drawbacks include its lack of homogeneity (mix- ture of elements of impairment and disability), as well as the interobserv- er reproducibility and sensitivity to change, which are not optimal. The Multiple Sclerosis Functional Composite (58), which was developed for therapeutic trials by a group of experts from several coun- tries, is a combination of three tests investigating motor function of the upper limbs, ambulation and cognitive functions. Every item is quantified from 0 to 4 according to the reported frequency of symp- toms involving fatigue during the previous four weeks, i. Cognitive impairment is frequent in multiple sclerosis (more than 60% of patients ten years after onset) and exhibit a rather character- istic pattern. Dysfunction of sustained attention, speed of informa- tion processing, and working memory are the earliest elements (60). Some patients ultimately develop subcortical dementia, which fortu- nately remains rare (61). The objectives of the evaluation differ de- pending on whether systematic detection, therapeutic studies, or patent cognitive complaints requiring appropriate management are involved. A standardized evaluation, the Brief Repeatable Battery was pro- posed by Rao in 1990 (63). The sensitivity is 70%, and its specificity is 94% in distinguishing patients with cognitive impairment among those followed for multiple sclerosis. In addition, frequent emotional problems, can result from difficul- ties in coping with the disease and/or from brain damage, particular- ly frontal lesions. The Barthel Index (16) is rarely used in multiple sclerosis in spite of its usefulness in predicting the duration of daily help (66). There are several well known disability assessment tools specifically designed for multiple sclerosis patients: The Hauser Ambulation Index (68) takes into account the quality of ambulation, the necessity of technical aids, walking speed (8 meters on level ground) and, for the patients in wheelchairs, independence for transfers. The evaluation of handicap and quality of life in multiple sclerosis is currently undergoing substantial expansion. The contents and construct validities, the in- ternal consistency, and the test-retest reproducibility of this scale are good. Although the length of time required for completion is relatively long, this battery shows good sensitivity to changes. Bladder dysfunction of patients with multiple sclerosis is one of the critical factors contributing to deterioration in the quality of life. The Qualiveen Questionnaire, developed for patients with spinal cord in- jury (75) and applied to multiple sclerosis patients, shows good va- lidity and discriminative sensitivity in this disease (76). The time required to complete this test is long and expertise is needed for the neurological examination (30 minutes for an experienced physician). Scales of dysarthria and dysphagia: One example is the adaptation of Frenchay Dysarthria Assessment (81), which is unfortunately on- ly available in French (82); another is the dysphagia scale proposed by Kennedy et al (83). The first to be used was the Hoehn and Yahr Scale (85) designed to determine the stage of progression of the disease. Two essential criteria of Parkinson’s disease are retained: a diagnostic criterion - unilaterality - and a course-related criterion - the onset of postural instability. This overall assessment has poor sensitivity, notably for therapeutic fol- low-up. The Schwab and England Scale (86) primarily assesses the level of dependence of patients to their family circle. The degree of overall au- tonomy is judged using values from 0% (maximum impairment) to 100% (normal). Numerous other generic or specific functional scales are proposed, some of which include quality-of-life concepts. In thirty- nine items, this scale provides a measurement of the quality of life in eight dimensions: mobility, activities of daily living, emotional well- being, stigma, social support, cognitive disorders, communication, and bodily discomfort.