By U. Pakwan. Colorado School of Mines.
A simple example using an episomal tetracycline gene as a target serves to illustrate the technique nicely cheap 400mg flagyl with visa. A chimeric oligonucleotide designed to mediate the correction is then transferred into the plasmid-containing bacterial cells generic 500 mg flagyl with amex. After a short recovery in medium containing tetracycline cheap flagyl 400mg online, the cells are grown for 16h in liquid medium order 250 mg flagyl. This experimental system addresses a series of impor- tant questions and concerns of genetic targeting: Is the conversion efﬁcient? Is the conversion stably transmitted to daughter cells and can the genetic change be prop- agated? The answers to all of these questions is, presumably, “yes,” when chimeric oligonu- cleotides are used in bacterial cells. Early attempts included increasing the length of the homology shared by the fragment and the genomic target. The topology of the targeting vehicle, usually a plasmid construct, was also modiﬁed but failed to improve the frequency of targeting speciﬁcity. Genomic Insertion To keep things in perspective, one must consider naturally occurring events that lead to insertions into the genome. The best example of this molecular process involves the integrative activity of viruses. As described earlier in this chapter and others, these viruses infect dividing cells at a high frequency but integrate randomly. Such observations have led to frustration among investigators hoping to use retroviruses for gene therapy. In some strategies, for example, precise integration would be helpful to achieve func- tional results. However, the central issue is that the cell does not naturally promote site-speciﬁc integration. Whether it is overwhelmed by the biological effort of the virus to integrate frequently or whether the enzymatic machinery driving homol- ogous insertion is naturally suppressed is not clear. This integrative event is catalyzed by the virally encoded Rep protein, an enzyme used to replicate the virus in the cell. Thus, a virally encoded protein, not a cellular enzyme, promotes site-speciﬁc targeting. Biochemical studies have shown that the Rep protein acts as a dimer, one subunit binding to the viral sequence and the other to the homologous viral-like sequence in the chromosome. The requirement for Rep binding sequences in both templates will clearly limit this approach. Hence two examples with naturally integrative elements (retroviruses and adenoassociated virus) have led investigators to conclude that homologous integration in mammalian cells is not a preferred or even a natural reaction. Gene Targeting: Gene Insertion or Gene Replacement in Mammalian Cells With this as a background, workers have attempted to translate the genetic obser- vations, and in some cases molecular tricks, found to work in lower eukaryotes or bacteria into the mammalian cell targeting arena. An early observation by yeast geneticists was that a double break in the homologous region of the targeting molecule elevated the frequency of site-speciﬁc integration. It had been widely accepted that double-stranded breaks promote homologous recombination even in mammalian cells, but the continual low frequency of speciﬁc events has persisted. To improve the frequency and develop reliable test systems, several strategies have emerged. After loxP sites are integrated into a mammalian genome, they can be used as integration sites for targeting vectors containing the transgene of choice and a compatible lox site, which is required for the speciﬁc “docking” effect mediated by Cre. On one level the frequency of integration at the “loxP site” is high and, on another level, the transgene can be excised since Cre works to promote both integration and excision. A similar system using a restriction endonuclease from yeast, known as I-SceI, can also be used (Fig. The recognition site for I-SceI is 18 base pairs in length, and thus the chances that multiple sites in the genome exist is fairly low. The major difference between I-SceI and Cre-lox is that in the I-SceI system, the target sequences are naturally present in the genome, albeit at rare frequency. The Cre recombinsase (transferase) and the Cre/lox vehicle are then added to the cells. In some cases, Cre may be expressed from a co-transfected plasmid containing the gene encoding Cre. By overexpressing Cre recombinase, the vector fragment or sequence can be exchanged in or out. The inefﬁciency of homologous recombination in mammalian cells may also be directly related to the low quality of gene transfer. Additional problems are fre- quent nonhomologous events, dependence of length of homologous target, and the lack of correlation between successful events and target copy number. To overcome at least one of these barriers, adenovirus vectors that cannot replicate have been developed. Since this virus infects essentially all of the cells, even a low-frequency event can be ampliﬁed if all of the cells receiving the vector undergo at least one homologous recombination reaction. The use of adenovirus to help in the gene transfer problem ampliﬁes a real problem for all efforts in the use of the homologous recombination; how does one insert the vector into enough cells to make a difference? The solution is to use micro- injection so that a vast majority of the cells receive the molecule. However, this procedure is highly labor intensive and essentially inappropriate to gene therapy strategies. Based on this information, workers have turned to the last alterable com- ponent of the gene targeting system: the cell. A large facet of successful gene targeting for in vitro studies is the culture condi- tions of the cells. It is possible that the achievement of high transfer efﬁciencies may be counterbalanced by the detrimental effects on nuclear metabolism. Simply insert- ing the vector into the cell is insufﬁcient; delivering it into the nucleus is the ulti- mate goal. Until liposomes or other delivery vehicles are able to target the vector to the speciﬁc site, this problem will persist. Such problems can be accentuated by using tissue culture cell lines that are consistently the same passage and the density at which the cells are plated can also inﬂuence the success rate of gene targeting events. Although these issues may seem mundane, they are critical to the develop- ment and assessment of the effectiveness of a particular vector prior to the move- ment of a technology forward with animal models or, ultimately, humans. In the mid- 1980s, several protocols were established wherein a speciﬁc, targeted gene could be rendered dysfunctional through the process of homologous recombination. Gene knock-outs in mice have become almost a routine step in the analysis of newly dis- covered gene function. It is almost a required step before the scientiﬁc community accepts the “deﬁnition” of a newly described gene. Although, in principal, generat- ing mouse knock-outs is routine, it is far from straightforward.
While on-call discount flagyl 250 mg with amex, residents are supervised by a Senior Staff Member discount flagyl 400 mg with visa, who is available at all times buy 200 mg flagyl otc, either via their office phone buy discount flagyl 250 mg on-line, pager or home phone. Scholarly Activities / Research Activities Residents are provided with continuous access to literature searching programs. The expectation is that residents will utilize the medical literature to find up-to-date information on their cases. It is further expected that residents will utilize the medical literature to help provide our clinical colleagues with up-to- date knowledge related to their cases. During sign-out and discussion of cases, the residents and teaching faculty discuss each case from a scholarly perspective. The resident and faculty discuss both normal and abnormal physiology and the mechanisms potentially responsible for creating the clinical findings observed. It is hoped that these discussions will foster an interest in research and the development of new knowledge. Residents are not only encouraged to become involved in research but technical, logistic, and economic support for such activities is available. Resident Evaluation Residents will be evaluated primarily on the performance of daily activities, particularly on evidence of their assimilation of the material present and their ability to apply it in a practical situation, namely, case Pathology Resident Manual Page 146 interpretation. Also, they will be evaluated on participation in required meetings, conferences, and their presentations. The residents are provided with continuous feedback on their performance during the rotation. Residents are evaluated on their demonstrated ability to provide informative consultation to the clinical service teams, their medical knowledge, their application of this knowledge to efficient/quality patient care, and their diagnostic, technical and observational skills. Residents are also evaluated on their interpersonal skills, professional attitudes, reliability, and ethics with members of the teaching faculty, peers, laboratory staff, and clinicians. They are further evaluated on their initiative in fostering quality patient care and use of the medical literature, as it relates to their assigned cases. Their timely completion of assigned interpretive reports is another component of the evaluation. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Bone Marrow Pathology): • Daily sign-out of bone marrow aspirations and biopsies: o Sign-out case with Pathology staff o Prior to sign out: Organize all slides for an individual case (blood smear, marrow aspirate smears, core biopsy, cell block, special stains) on a single slide tray dedicated for that case Obtain all pertinent information for each case, including previous histologic material, laboratory data, and clinical data Write-up each case and establish a diagnosis • Maintain an organized marrow sign-out room. The first approach is to ask the Wet Hematology resident, provided that the Wet Hematology resident has experience in performing bone marrow biopsies. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature. Distinguish between urate and calcium pyrophosphate crystals, using polarized light. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature. Duties and Responsibilities of Residents • Be present in the laboratory for significant periods of time to allow exposure to routine laboratory work. Duties and Responsibilities of Residents • Be present in the laboratory for significant periods of time to allow exposure to routine laboratory work. Resident Evaluation • The residents must show active participation in the laboratory. Pathology Resident Manual Page 172 • The resident must be reliable and responsible for clinical case presentations. Manual of Clinical Microbiology, 10 Edition, American Society for Microbiology, Washington, D. Clinical Immunology: Principles and Laboratory Diagnosis, 2 Edition, Lippincott-Raven, 1997 3. Management and informatics activities are not limited to this single rotation, but should be considered to span the entire length of the residency. The activities are present in both the laboratory and administrative areas of the Department. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature. Compare and contrast the structure of Pathology Resident Manual Page 177 differing practice settings (e. Utilize these methodologies to select and validate new diagnostic tests and analytical methods. Pathology Resident Manual Page 180 • Understand the essential elements of choosing a reference laboratory. Interpersonal and Communication Skills • Understand how to conduct an interview for a new employee. Systems-Based Practice • Understand the differences between different forms of professional practice. Medical Informat ics: Computer Applications in Health Care and Biomedicine, 2nd ed. Duties and Responsibilities Each resident is expected to: • Become familiar with the schedule of the formal activities cited above and to participate on a regular basis. The activities are present in both the medical and administrative areas of the Department. During the first three months of starting the residency, a session is held with the Administrative Lab Director. The Misys modules installed include Anatomic Pathology (CoPath module), General Laboratory and Blood Bank. Each laboratory department uses worksheets or pending logs to display outstanding tests. The rotation aims to expose the resident to various aspects of management and service work in an integrated clinical pathology laboratory. Residents will assume graded responsibility in subsequent months on this rotation. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature. Distinguish between urate and calcium pyrophosphate crystals, using polarized light. Resident Evaluation • The resident must show active participation in the functions of the clinical pathology laboratory. Objectives: Learning Evaluation Activities Activities Demonstrate the ability to critically assess the scientific literature.
Surgical intervention or stent placement is nec- Lupus Plasmapheresis Study Group: rationale and updated essary in patients with renovascular hypertension or interim report 400mg flagyl amex. Haematopoietic stem cell gene therapy to treat autoimmune Medicine (Baltimore) 52:535–61 disease 200mg flagyl with visa. Chin Med J (Engl) cell antibodies mediate enhanced leukocyte adhesion 115:705–9 to cytokine-activated endothelial cells through a novel 39 purchase flagyl 200mg line. Circulation of good-prognosis polyarteritis nodosa and Churg-Strauss 90:1855–60 syndrome: comparison of steroids and oral or pulse cyclo- 41 buy flagyl 250mg. Glicklich D, Acharya A (1998) Mycophenolate mofetil Care Med 173:180–187 therapy for lupus nephritis refractory to intravenous cyclo- 44. Am J Kidney Dis 32:318–22 granulomatosis: long-term follow-up of patients treated 29. Arthritis Rheum 42:2666–2673 phosphamide in the treatment of generalized Wegener’s 46. Am J riority of steroids plus plasma exchange to steroids alone Med 67:941–7 in the treatment of polyarteritis nodosa and Churg-Strauss 48. Hellmich B, Lamprecht P, Gross W (2006) Advances in the Heart J 93:94–103 therapy of Wegener’s granulomatosis. Ann Intern vasculitis and renal involvement: A prospective, randomized Med 116:488–98 study. Arthritis Rheum 58:308–17 yarteritis: presence of anti-endothelial cell antibodies and 55. Clin Lupus Erytmatosus, Anaphylactoid Purpura and Vasculitis Exp Immunol 85:14–9 Syndromes. Arthritis Rheum 54:2970–82 pilot trial comparing cyclosporine and azathioprine for 75. Niaudet P, Habib R (1998) Methylprednisolone pulse ther- Ann Intern Med 75:165–71 apy in the treatment of severe forms of Schonlein- Henoch 77. Pediatr Nephrol 12:238–43 renal prognosis of Henoch-Schönlein Purpura in an unse- 61. The Wegener’s Granulomatosis Etanercept Trial Research therapy on rapidly progressive type of Henoch-Schonlein G (2005) Etanercept plus standard therapy for Wegener’s nephritis. Takayasu’s arteritis: correlations of their titers and isotype Scand J Rheumatol 33:423–7 distributions with disease activity. Robbins S, Cotran R, Kumar V, Inflammation - The with special reference to renal involvement. Two months posttransplant, graft dysfunction developed and was found to be caused by obstruction of the transplant ure- more definitive intervention, the ureteral stent was ter at the level of the bladder anastomosis. A ureteral removed during the cystoscopy, and the patient was stent was placed, graft function stabilized (serum cre- monitored closely for recurrence of graft dysfunction, atinine 0. With this support, the patient stabilized and general categorization according to transplant status eventually recovered, including his graft function, and introduced above. Under may affect the patient’s transplant candidacy signifi- all of these circumstances, renal dysfunction can occur, cantly, either by presenting a potential contraindication typically requiring complex management tailored to the to the desired nonrenal transplantation or by establishing specific needs of the individual patient. Some patients with renal dysfunction prior to non- highly multidisciplinary fashion, usually codirected by renal organ transplantation may be expected to recover a combination of intensivists, pediatric subspecialists, kidney function after nonrenal transplantation, likely and transplant surgeons and their teams. Such decisions young recipients of a preemptive transplant from a and plans are examples for the aforementioned complex living adult donor, this complication also appears to multidisciplinary, individualized, and communicative be driven by dramatic decreases in serum osmolality management approach for these patients and require associated with rapid clearance of uremic toxins from thorough consideration of medical prognosis, quality of the circulation when renal graft function is excellent life implications, and other, e. Even in older and bigger recipients, the frequency and volume of urine output measurements and replace- 18. Recovery of tubular abilities to concentrate the urine and reabsorb sodium usually takes several days, over which urine output replacement is gradu- 18. Of critical importance is the realization that the hourly urine output may actu- Table 18. Generally, circumstances, particularly when an adult allograft immunosuppressive therapy is in constant evolution is placed into an infant. This creates a tremendous to achieve the best possible antirejection prophylaxis Table 18. In this context, it has become Hypertension frequently occurs or worsens in the quite clear that immunosuppressive protocols cannot immediate posttransplant setting for several reasons, be administered in a one size fits all fashion: First-time including liberal fluid management (see above) and Caucasian recipients of a living donor kidney who have treatment with high doses of corticosteroids. While no evidence of presensitization appear to require less mild blood pressure elevations above the recipi- powerful antirejection prophylaxis than recipients of a ent’s pretransplant range may be temporarily desir- repeat transplant, especially one from a deceased donor, able to enhance perfusion of the new allograft, more recipients with evidence of presensitization, or recipi- pronounced hypertension, especially if it is causing ents who are African-American . In this set- recent discovery of genetic polymorphisms and related ting, calcium channel antagonists are particularly safe phenomena affecting drug metabolism and exposure and effective, although attention needs to be paid to [7, 15] and immunological responsiveness  further the interference of some of these agents, particularly undermines the concept of a unified immunosuppres- verapamil, diltiazem, amlodipine, and nicardipine sive approach. Once transplant function programs to adapt flexible protocols that can be tailored has stabilized, the same group of agents may also be to each recipient’s perceived risk profile. Prophylaxis against bacterial, viral, and fungal patho- com/study/ped/annlrept/annlrept. Nonetheless, additional guidance in the selec- provided perioperatively to prevent wound infections tion of pediatric immunosuppressive regimens is also and then transitioned to a prophylactic regimen against derived from adult studies and from local practice and urinary tract infections and pneumocystis carinii. Specific guidelines have been developed for antiviral A typical protocol to be used initially in pediat- prophylaxis in the posttransplant setting . Accordingly, a number carries a substantial long-term risk of nephrotoxicity of centers also recycle the full spectrum of infection [23, 28]. Similar principles apply to nonrenal transplant prophylaxis during and after episodes of acute rejection recipients [27, 31, 41, 51]. Many centers there- Transplantation fore perform a Doppler ultrasonographic evaluation or a nuclear scan of the transplant immediately after skin Gastrointestinal Prophylaxis closure or upon arrival in the postoperative care unit, Gastrointestinal prophylaxis against steroid-associated at least if there is no sufficient urine output attributable gastritis and ulcer disease is typically given in the form to the transplant. At our center, still have their native, oftentimes urine-producing, kid- recipients are tried off these agents once they are neys at the time of transplantation, making the precise taking all their medicines by mouth and if they are free determination of the source of urine output – i. Prophylaxis Against Thrombosis If blood flow to the transplant is adequate, acute Graft thrombosis is a significant cause of pediat- tubular necrosis should be suspected as alternative ric transplant loss [49, 56]. Risk factors include cause of initial nonfunction, especially in transplants hypercoagulopathy (e. In recipients who are not at states), antiphospholipid antibodies (seen in 30–50% particularly increased immunological risk, hyperacute of patients with systemic lupus erythematosus), prior rejection is very unlikely. Accordingly, hyper- coagulability should be corrected before the actual Delayed-Onset Graft Dysfunction transplant procedure whenever possible. Alternatively, In grafts with initially acceptable urine production consideration needs to be given to the prescription but a subsequent decrease in output, additional pos- of anticoagulation during and after the transplant, sibilities need to be considered. Both Initial Nonfunction of these complications can obviously also occur after Graft dysfunction immediately posttransplant is sug- transplantation of nonrenal organs. Accordingly, initial nonfunction requires imme- responses: Especially in presensitized recipients, acute Table 18. Goebel rejection can not only be cellular but also antibody- by a blood clot, and urinary leakage, e.