Model-guided design of mammalian genetic programs.

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

Robotically assisted laparoscopic transhiatal esophagectomy.

BACKGROUND: Esophagectomy is a technically demanding operation with high procedure-related morbidity and mortality rates. Minimally invasive techniques were introduced in the late 1980s in an effort to decrease the invasiveness of the procedure. Data concerning the use of robotic systems for esophageal cancer are scarce in the literature. The goal of this report is to describe the authors' early experience using robotically assisted technology to perform transhiatal esophagectomy (RATE). METHODS: Between September 2001 and May 2004, 18 patients underwent RATE at the authors' institution. A retrospective review of prospectively collected data was performed. Gender, age, postoperative diagnosis, operative time, conversion rate, blood loss, hospital stay, length of the follow-up period, and complications were assessed. RESULTS: At the authors' institution, 18 patients underwent RATE, including 16 men (89%), with a mean age of 54 years (range, 41-73 years). The RATE procedure was completed for all 18 patients (100%). The mean operative time was 267 +/- 71 min, and estimated blood loss was 54 ml (range, 10-150 ml). The mean intensive care unit stay was 1.8 days (range, 1-5 days), and the mean hospital stay was 10 days (range, 4-38 days). A total of 12 perioperative complications occurred for 9 patients, including 6 anastomotic leaks, 1 thoracic duct injury, 1 vocal cord paralysis, 1 pleural effusion, and 2 atrial fibrillations. Anastomotic stricture was observed in six patients. There were no perioperative deaths. Pathologic examination of the surgical specimen yielded an average of 14 lymph nodes per patient (range, 7-27). During the mean follow-up period of 22 +/- 8 months, 2 patients died, 2 were lost to follow-up evaluation, 3 had recurrence, and 11 were disease free. CONCLUSION: The current study shows that RATE, with its decreased blood loss, minimal cardiopulmonary complications, and no hospital mortality, represents a safe and effective alternative for the treatment of esophageal adenocarcinoma.

Laparoscopic Heller myotomy for achalasia facilitated by robotic assistance.

BACKGROUND: Laparoscopic Heller myotomy is the standard operation for achalasia. The incidence of esophageal perforation is approximately 5% to 10%. Data about the safety and utility of robotically assisted Heller myotomy (RAHM) are scarce. The aim of this study was to assess the efficacy and safety of RAHM for the treatment of esophageal achalasia. METHODS: From a prospectively maintained database, demographic data, symptoms, esophagograms, manometries, and perioperative data from all the RAHMs performed between September 2002 and February 2004 were analyzed. RESULTS: A total of 54 patients underwent RAHM, including 26 men. The mean age of these patients was 43 years (range, 14-75 years). Dysphagia was present in 100% of the patients. Of the 54 patients, 26 (48%) had undergone previous treatment including pneumatic dilation (17 patients), Botox injections (4 patients), or both of these treatments (5 patients). The dissection was performed laparoscopically, and the myotomy was performed with robotic assistance. The operative time, including the robot setup time, averaged 162 min (range, 62-210 min). Blood loss averaged 24 ml. No mucosal perforations were observed. The hospital length of stay was 1.5 days. There were no deaths. At 17 months, 93% of the patients had relief of their dysphagia. CONCLUSIONS: The findings showed RAHM to be safe and effective, with a 0% incidence of perforation and relief of symptoms for 91% of the patients.

Lower esophageal sphincter analysis using computerized manometry in patients with chagasic megaesophagus.

Due to the introduction of computer technology into manometry laboratories, three-dimensional manometric images of the lower esophageal sphincter can be constructed based on radially oriented pressures, a method termed 'computerized axial manometry.' Calculation of the sphincter pressure vector volume using this method is superior to standard manometric techniques in assessing lower esophageal sphincter function in patients with gastroesophageal reflux disease and idiopathic achalasia. Despite similarities between idiopathic achalasia and chagasic esophagopathy found using clinical, radiological, and manometric studies, controversy around lower esophageal sphincter pressure persists. The goal of this study was to analyze esophageal motor disorders in Chagas' megaesophagus using computerized axial manometry. Twenty patients with chagasic megaesophagus (5 men, 15 women, and average age 50.1 years, range 17-64) were prospectively studied. For three-dimensional imaging construction of the lower esophageal sphincter, a low-complacency perfusion system and an eight-channel manometry probe with four radial channels placed in the same level were used. For probe traction, the continuous pull-through technique was used. Results showed that the lower esophageal sphincter of patients with chagasic megaesophagus have significantly elevated pressure, length, asymmetry, and vector volumes compared to those of normal volunteers (P < 0.05). Aperistalsis of the esophageal body waves was observed in all patients and contraction amplitude was lower than that in normal patients. We conclude that patients with chagasic megaesophagus have hypertonic lower esophageal sphincter and aperistalsis of the esophageal body.

Effect on treatment outcome of coinfection with SEN viruses in patients with hepatitis C.

The newly discovered SEN D and SEN H viruses are transmitted parenterally and can cause post-transfusion hepatitis. We assessed whether coinfection of patients with chronic hepatitis C and SEN D or SEN H correlates with the outcome of treatment with interferon and ribavirin. Of 31 patients with hepatitis C studied, six were positive for SEN D and seven for SEN H (one was positive for both). All of those positive for SEN D and five of those positive for SEN H failed to respond to therapy. Overall response (RNA titre and alanine aminotransferase concentration after treatment) was lower in SEN-infected patients than uninfected patients (p=0.025). We conclude that coinfection with SEN viruses is frequent in chronic hepatitis C patients and might adversely affect the outcome of treatment with interferon and ribavirin.

SEN virus infection and its relationship to transfusion-associated hepatitis.

SEN virus (SEN-V) is a recently identified single-stranded, circular DNA virus. Two SEN-V variants (SENV-D and SENV-H) were assayed by polymerase chain reaction (PCR) to investigate their role in the causation of transfusion-associated non-A to E hepatitis. The incidence of SEN-V infection after transfusion was 30% (86 of 286) compared with 3% (3 of 97) among nontransfused controls (P < .001). Transfusion risk increased with the number of units transfused (P < .0001) and donor-recipient linkage for SEN-V was shown by sequence homology. The prevalence of SEN-V in 436 volunteer donors was 1.8%. Among patients with transfusion-associated non-A to E hepatitis, 11 of 12 (92%) were infected with SEN-V at the time of transfusion compared with 55 of 225 (24%) identically followed recipients who did not develop hepatitis (P < .001). No effect of SEN-V on the severity or persistence of coexistent hepatitis C virus (HCV) infection was observed. In 31 infected recipients, SEN-V persisted for greater than 1 year in 45% and for up to 12 years in 13%. SEN-V-specific RNA (a possible replicative intermediate) was recovered from liver tissue. In summary, SENV-D and -H were present in nearly 2% of US donors, and were unequivocally transmitted by transfusion and frequently persisted. The strong association of SEN-V with transfusion-associated non-A to E hepatitis compared with controls raises the possibility, but does not establish that SEN-V might be a causative agent of posttransfusion hepatitis. The vast majority of SEN-V-infected recipients did not develop hepatitis.

Serum amylase and lipase elevation is associated with intracranial events.

Serum amylase and lipase elevation has been observed in trauma patients and patients with traumatic intracranial bleeding. However, the causes of this elevation have not been clearly elucidated. A further question remains as to whether other intracranial events are associated with such enzyme elevation as well. We retrospectively reviewed 75 patients consecutively admitted to Cook County Hospital Neurosurgical Intensive Care Unit over a 3-month period for trauma, infection, tumor, or other space-occupying lesions with an unstable condition or neurological deficit. Eleven patients (15%) had elevated amylase and lipase levels. The patients were divided into two groups: Group I (n = 64) had normal and Group II (n = 11) had raised amylase and lipase levels [amylase 402 +/- 444 U/L with normal < or = 125 U/L and lipase 474 +/- 313 U/L with normal < or = 55 U/L]. All Group II patients suffered an intracranial event. Twenty-four Group I (38%) and 10 Group II (91%) patients required craniotomy (P < 0.01). No patients had clinical or radiographic evidence of pancreatitis. In summary, intracranial events are associated with serum amylase and lipase elevation probably through centrally activated pathways. Because of the lack of diagnostic value, routine pancreatic enzyme monitoring should not be performed in this patient population.

Hypocaloric parenteral nutrition support in elderly obese patients.

Hypocaloric nutritional support has been successfully used to achieve positive nitrogen balance in obese patients. However, advanced age is associated with changes in substrate metabolism. To evaluate the efficacy of this practice in elderly patients, we retrospectively reviewed 30 obese patients requiring parenteral nutrition support. Total caloric requirements (TCR) were calculated on the basis of the Harris-Benedict equation. Patients were given a minimum of 1.5 g/kg/day protein, 75 per cent of TCR when current body weight was >120 and < or =150 per cent of ideal body weight (IBW), and 60 per cent of TCR if current body weight was >150 per cent of IBW. The patients were divided into two groups: Group I (N = 18) patients were age <60 and Group II (N = 12) patients were > or =60 years of age. Only one of the Group I patients (143.2 +/- 25.7% IBW, received 18.2 +/- 3.7 kcal/kg/day and 1.6 +/- 0.4 g/kg/day protein), but five of the Group II patients (141.3 +/- 14.4% IBW, received 18.3 +/- 2.6 kcal/kg/day and 1.7 +/- 0.3 g/kg/day protein), had negative nitrogen balance (P = 0.025). Apparently, elderly obese patients have limited capacity to mobilize their fat stores as energy sources and may continue to undergo protein catabolism with hypocaloric high-protein nutrition support. Therefore, this form of nutritional support should be used with caution in elderly obese patients.

Intraoperative heparin in addition to postoperative low-molecular-weight heparin for thromboprophylaxis in total knee replacement.

The administration of heparin during operation has been reported to enhance the efficacy of thromboprophylaxis in patients undergoing total hip replacement. We have performed a small pilot study in which intraoperative doses of heparin were given in addition to the usual postoperative thromboprophylaxis with enoxaparin in 32 patients undergoing total knee replacement. The primary endpoint was deep-vein thrombosis (DVT) as demonstrated by bilateral venography on 6 +/- 2 days after operation. Sixteen patients developed DVT; in two the thrombosis was proximal as well as distal and in one the occurrence was bilateral. There was one major haemorrhage. These results are similar to those obtained with the use of postoperative thromboprophylaxis with enoxaparin alone. They do not provide support for the initiation of a larger randomised trial of this approach to management.

Parietal cell vagotomy versus vagotomy-antrectomy: ulcer surgery in the modern era.

Patients with peptic ulcer occasionally develop complications that require surgical intervention, despite the advances in medical treatment and changes in the natural history of disease. The clinical surgeon must make a decision about performing "selective vagotomy antrectomy versus highly selective vagotomy," based on the information discussed herein. The goals for operative treatment remain safe correction of the presenting problem, avoidance of perioperative morbidity and mortality, and freedom from disabling postoperative side effects. This paper addresses broad aspects of the details of surgical interventions; because most operative procedures are performed in urgent circumstances in patients who often have a variety of conditions, it is not surprising that there is no best operation suited to every complication of ulcer.

Laparoscopic myotomy in achalasia: intraoperative evidence for myotomy of the gastric cardia.

The myotomy performed for achalasia of the esophagus should divide all of the constricting, diseased muscular elements that obstruct the esophagogastric junction (EGJ). Whether the disease process includes proximal gastric as well as esophageal components is as yet unclear, but anatomic evidence complemented by clinical data suggest that the disease process does not end at the evanescent and poorly defined EGJ. Clinical reports from enthusiastic proponents of a particular operative approach for achalasia have not been illuminating in this regard, because all patients are improved to some degree post-operatively, and there are no objective parametric standards for the evaluation of swallowing function. This study reports a series of patients in whom endoscopic viewing was used to judge the adequacy of myotomy after 'esophageal' myotomy. The question posed by this study was, 'Does esophageal myotomy remove all constricting elements at the gastroesophageal junction?' Laparoscopic myotomy was performed in 48 patients with a diagnosis of achalasia; these patients are the most recent in a total cohort of 72 patients operated upon for achalasia during the past 20 years. Myotomy was begun on the esophagus, and extended to the esophagogastric junction; anatomic landmarks, including the appearance of submucosal veins, guided the initial dissection. Intraoperative endoscopy was then performed to determine whether there was residual constriction of the channel between the esophagus and stomach; if so, myotomy was extended onto the gastric cardia until visual evidence of obstruction had disappeared. All patients had either Toupet fundoplication or Dor fundoplication after myotomy. There were obvious constricting elements distal to the gastroesophageal junction in 90% of the patients. These patients required extension of the myotomy onto the stomach for an average of 15 mm. All but one patient had improved swallowing post-operatively. Eight patients required 'stretch' of the distal esophagus/cardia within the first year post-operatively; one patient was reoperated for fibrous scar obstruction of the distal esophagus. Esophageal myotomy limited to the esophageal muscle does not remove all constricting elements at the gastroesophageal junction; as a result, the extended myotomy must be complemented by an antireflux procedure during operations for achalasia.

Evolution of a simian immunodeficiency virus pathogen.

Analysis of disease induction by simian immunodeficiency viruses (SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since they failed to induce disease within 1 year postinoculation in any inoculated animal. Here we report the natural history of infection with BK28 and BK44 in inoculated rhesus macaques and efforts to increase the pathogenicity of BK28 through genetic manipulation and in vivo passage. BK44 infection resulted in no disease in four animals infected for more than 7 years, whereas BK28 induced disease in less than half of animals monitored for up to 7 years. Elongation of the BK28 transmembrane protein (TM) coding sequence truncated by prior passage in human cells marginally increased pathogenicity, with two of four animals dying in the third year and one dying in the seventh year of infection. Modification of the BK28 long terminal repeat to include four consensus nuclear factor SP1 and two consensus NF-kappaB binding sites enhanced early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from immunodeficiency disease (1.5 years after infection) resulted in a consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains identified to date. To determine whether the diverse viral quasispecies that evolved during in vivo passage was required for pathogenicity or whether a more virulent virus variant had evolved, we generated a molecular clone composed of the 3' half of the viral genome derived from the in vivo-passaged virus (H824) fused with the 5' half of the BK28 genome. Kinetics of disease induction with this cloned virus (BK28/H824) were similar to those with the in vivo-passaged virus, with four of five animals surviving less than 1.7 years. Thus, evolution of variants with enhanced pathogenicity can account for the increased pathogenicity of this SIV strain. The genetic changes responsible for this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have been multiple and dispersed, including elongation of the TM and Nef coding sequences; changes in RNA splice donor and acceptor sites, TATA box sites, and Sp1 sites; multiple changes in the V2 region of SU, including a consensus neutralization epitope; and five new N-linked glycosylation sites in SU.

Trovafloxacin in the treatment of intra-abdominal infections: results of a double-blind, multicenter comparison with imipenem/cilastatin. Trovafloxacin Surgical Group.

BACKGROUND: Trovafloxacin, a new broad-spectrum fourth-generation quinolone, has in vitro activity against most gram-negative and gram-positive anaerobes and aerobes. Trovafloxacin is available as both an intravenous formulation, alatrofloxacin, and a single daily oral tablet. Excellent tissue pharmacokinetics and oral bioavailability suggest usefulness in the treatment of complicated intra-abdominal infections. Thus, the efficacy of alatrofloxacin followed by oral trovafloxacin was compared with the standard regimen of intravenous imipenem/cilastatin followed by oral amoxicillin/clavulanic acid in this prospective, multicenter, double-blind trial. METHODS: Patients were randomized to receive either 300 mg alatrofloxacin daily followed by 200 mg oral trovafloxacin daily or 1 g imipenem/cilastatin intravenously thrice daily followed by 500 mg oral amoxicillin/clavulanic acid thrice daily for up to 14 days following surgical intervention of a documented intra-abdominal infection. Efficacy was assessed at the end of therapy and at follow-up (day 30). RESULTS: At the end of the study, cure or improvement occurred in 83% (129/156) and 84% (127/152) of clinically evaluable patients in the trovafloxacin and comparative groups, respectively. Pathogen eradication rates, adverse-event profiles, and significant laboratory abnormalities were comparable between groups. CONCLUSION: Intravenous alatrofloxacin with or without oral trovafloxacin was as effective as intravenous imipenem/cilastatin followed by oral amoxicillin/clavulanic acid in complicated intra-abdominal infections.

Basic considerations in gastroesophageal reflux disease.

The cause of foregut symptoms is often quite uncertain until a comprehensive evaluation has been performed. The critical elements of this evaluation include historic, radiographic, endoscopic, and physiologic data, and most importantly, the insight of a mature diagnostician. Patients who are not evaluated in a comprehensive way are at risk for serious postoperative problems; surgeons who perform interventions without appropriate diagnostic support may have to deal with these unhappy patients. In the long run, a complete workup provides the guidance for treatment and is cost-effective.

Carcinoma involving the gallbladder in elderly patients presenting with acute cholecystitis.

BACKGROUND: The unexpected intraoperative finding of a cancerous gallbladder has become particularly problematic, because cancer recurs rapidly after laparoscopic cholecystectomy. It would be desirable to identify the patients of greatest risk for gallbladder cancer before operation. After several elderly patients presenting with acute cholecystitis were found to have gallbladder cancer, we performed the following study. METHODS: Records of patients (60 years of age or older, 1987 to 1995) with an admitting diagnosis of acute cholecystitis and symptoms including right upper quadrant pain, nausea, vomiting, fever, and leukocytosis were reviewed. RESULTS: Eighty patients were included in the study. Carcinoma involving the gallbladder was found in seven patients; six had primary and one had metastatic carcinoma. The 73 patients without cancer underwent cholecystectomy. The differences between the noncancer and cancer patients included age (68 +/- 7 versus 74 +/- 8 years, p < 0.05), total bilirubin (mg/dl, 1.5 +/- 1.5 versus 3.7 +/- 3.4, p < 0.01), alkaline phosphatase (IU/L, 179 +/- 132 versus 369 +/- 226, p < 0.01), and aspartate aminotransferase (IU/L, 77 +/- 93 versus 158 +/- 157, p < 0.05). CONCLUSIONS: Additional work-up and open cholecystectomy should be considered in elderly patients presenting with apparent acute cholecystitis, especially when liver functions are abnormal.

Cholestasis in patients with acquired immunodeficiency syndrome: a surgeon's perspective.

Cholestasis is a common finding in patients with acquired immunodeficiency syndrome. The underlying causes may be related to intrahepatic processes, cholecystitis, papillary stenosis, or sclerosing cholangitis. Published reports of hepatobiliary diseases in patients with acquired immunodeficiency syndrome are reviewed. The etiological factors are considered, and available therapeutic approaches are discussed. Hepatic causes of cholestasis indicate poor prognosis, and effective treatments are not yet available. Cholecystitis, often acalculous in origin, remains a surgical disease. Endoscopic sphincterotomy appears to give good results in the treatment of papillary stenosis. Although a standard approach to sclerosing cholangitis has not been established, a trial of appropriate antibiotics may be beneficial. Frequently, the cause of cholestasis is multifactorial in these patients; thus, a coherent therapeutic approach is essential for optimal clinical results.