Dnmt3a regulates myeloproliferation and liver-specific expansion of hematopoietic stem and progenitor cells.

DNA methyltransferase 3A (DNMT3A) mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here, we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell-autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo.

Laparoscopically monitored colonoscopic polypectomy: an established form of endoluminal therapy for colorectal polyps.

BACKGROUND: Benign polyps, the most common disorders of the colon, are considered by many to be premalignant lesions. Colonoscopy is widely used to remove the majority of these polyps. However, a variety of "difficult polyps" are not accessible for colonoscopic removal because of their location and size, the tortuosity of the colon, or the complexity of the lesion (sessile vs pedunculated). In the past, a formal segmental resection usually was suggested for these difficult polyps. METHODS: For 110 patients with a median age of 73 years, a total of 149 polyps were removed as follows: 88 from the right colon, 18 from the transverse colon, 10 from the left colon, and 33 from the rectosigmoid colon. Pathologic evaluation showed adenomatous polyps in 13 patients (11.81%), hyperplastic polyps in 1 patient (0.9%), adenocarcinomas in 10 patients (9.09%), carcinoma in situ in 1 patient (0.9%), and adenomas in the remaining patients (tubulovillous, 40.18%; villous, 19.31%, and tubular, 17.77%). All the specimens were evaluated for margins and depth of resection. RESULTS: The median size of the polyps was 2.30 cm (range, 0.2-6 cm). The average hospital stay was 1.14 days, with a liquid diet started 6 h postoperatively. Mild abdominal pain/trocar-site pain was the most common complaint. The patients were followed with colonoscopy 6 months postoperatively and yearly thereafter. CONCLUSIONS: A combined endoscopic-laparoscopic approach provides a valid alternative for treating difficult colonic polyps and eliminating the morbidity of a segmental resection. This approach seems to be safe and effective.

Preliminary results of a two-layered prosthetic repair for recurrent inguinal and ventral hernias combining open and laparoscopic techniques.

The use of prosthetic mesh has become the standard of care in the management of hernias because of its association with a low rate of recurrence. However, despite its use, recurrence rates of 1% have been reported in primary inguinal repair and rates as high as 15% with ventral hernia repair. When dealing with difficult recurrent hernias, the two-layer prosthetic repair technique is a good option. In the event of incarcerated or strangulated hernias, however; placement of prosthetic material is controversial due to the increased risk of infection. The same is true when hernia repairs are performed concurrently with potentially contaminated procedures such as cholecystectomy, appendectomy, or colectomy. The purpose of this study is to report our preliminary results on the treatment of recurrent hernias by combining laparoscopic and open techniques to construct a two-layered prosthetic repair using a four ply mesh of porcine small intestine submucosa (Surgisis, Cook Surgical, Bloomington, IN, USA) in a potentially infected field and a combination of polypropylene and ePTFE (Gore-Tex, W.L. Gore and Associates, Flagstaff, AZ, USA) in a clean field. From September 2002 to January 2004, nine patients (three males and six females) underwent laparoscopic and open placement of surgisis mesh in a two layered fashion for either recurrent incisional or inguinal hernias in a contaminated field. A total of eight recurrent hernia repairs were performed (five incisional, three inguinal) and one abdominal wall repair after resection of a metastatic tumor following open colectomy for colon carcinoma. Six procedures were performed in a potentially contaminated field (incarcerated or strangulated bowel within the hernia), two procedures were performed in a contaminated field because of infected polypropylene mesh, and one was in a clean field. Mean patient age was 56.4 years. The average operating time was 156.8 min. Operative findings included seven incarcerated hernias (four incisional and three inguinal), one strangulated inguinal hernia, and one ventral defect after resection of an abdominal wall metastasis for a previous colon cancer resection. In two of the cases, there was an abscess of a previously placed polypropylene mesh. All procedures were completed with two layers of mesh (eight cases with surgisis and one with combination of polypropylene/ePTFE). Median follow up was 10 months. Complications included two seromas, one urinary tract infection, two cases of atelectasis and one prolonged ileus. There were no wound infections. The average postoperative length of stay was 7.8 days. There have been no mesh-related complications or recurrent hernias in our early postoperative follow-up period. The use of a new prosthetic device in infected or potentially infected fields, and the two-layered approach shows promising results. This is encouraging and provides an alternative approach for the management of difficult, recurrent hernias.

Use of porcine small intestinal submucosa as a prosthetic device for laparoscopic repair of hernias in contaminated fields: 2-year follow-up.

BACKGROUND: Surgisis is a new four- or eight-ply bioactive, prosthetic mesh for hernia repair derived from porcine small intestinal submucosa (SIS). It is a naturally occurring extracellular matrix, which is easily absorbed, supports early and abundant new vessel growth, and serves as a template for the constructive remodeling of many tissues. As such, we believe that Surgisis mesh is ideal for use in contaminated or potentially contaminated fields in which ventral, incisional, or inguinal hernia repairs are required. METHODS: From November 2000-May 2003, 53 patients (23 male, 30 female) underwent placement of Surgisis mesh for a variety of different hernia repairs. A total of 58 hernia repairs were performed in our patient population. Twenty procedures (34%) were performed in a potentially contaminated setting (i.e., with incarcerated/strangulated bowel within the hernia or coincident with a laparocopic cholecystectomy/colectomy). Thirteen repairs (22%) were performed in a grossly contaminated field, including one in which an infected polypropylene mesh from a previous inguinal hernia repair was replaced with Surgisis mesh and one in which dead bowel was discovered within the hernia sac. Median follow-up is 19 months with a range of 1-30 months. RESULTS: Of the 58 total repairs, there was one wound infection complicated by enterocutaneous fistula in a patient originally operated on for ischemic bowel. The fistula was in a location independent of the Surgisis mesh. There have been no mesh-related complications or recurrent hernias in our early postoperative follow-up period. CONCLUSIONS: Surgisis mesh appears to be a promising new prosthetic material for hernia repair and appears to function well, especially in contaminated or potentially contaminated fields. Obviously, long-term follow-up is still required.

Laparoscopic diagnosis and treatment of intestinal obstruction.

BACKGROUND: Intestinal obstruction is a common reason for general surgical referral. The traditional approach has been conservative management, followed by laparotomy if conservative measures are unsuccessful. However, with the advent of minimally invasive surgery, the need for laparotomy for this common problem is being challenged. METHODS: From May 1991 to April 2001, 167 patients underwent laparoscopy for diagnosis and/or treatment of intestinal obstruction. Average patient age was 62 years (range, 21-98). The site of obstruction was the stomach in seven patients, small bowel in 116 patients, and colon in 44 patients. RESULTS: Laparoscopy successfully diagnosed the site of obstruction in all patients. In addition, 154 patients (92.2%) were successfully treated laparoscopically without conversion to laparotomy. Both intraoperative and postoperative complication rates were low (3.5 and 18.6%, respectively) and compared favorably with those of published reports. CONCLUSIONS: Intestinal obstruction can be approached safely and effectively by laparoscopy with the intent not only to correctly diagnose the patient but also to render treatment.

Laparoscopic ventral and incisional hernia repair: an 11-year experience.

Incisional hernias develop in 2%-20% of laparotomy incisions, necessitating approximately 90000 ventral hernia repairs per year. Although a common general surgical problem, a "best" method for repair has yet to be identified, as evidenced by documented recurrence rates of 25%-52% with primary open repair. The aim of this study was to evaluate the efficacy and safety of laparoscopic ventral and incisional herniorrhaphy. From February 1991 through November 2002, a total of 384 patients were treated by laparoscopic technique for primary and recurrent umbilical hernias, ventral incisional hernias, and spigelian hernias. The technique was essentially the same for each procedure and involved lysis of adhesions, reduction of hernia contents, closure of the defect, and 3-5 cm circumferential mesh coverage of all hernias. Of the 384 patients in our study group, there were 212 females and 172 males with a mean age of 58.3 years (range 27-100 years). Ninety-six percent of the hernia repairs were completed laparoscopically. Mean operating time was 68 min (range 14-405 min), and estimated average blood loss was 25 mL (range 10-200 mL). The mean postoperative hospital stay was 2.9 days and ranged from same-day discharge to 36 days. The overall postoperative complication rate was 10.1%. There have been 11 recurrences (2.9%) during a mean follow-up time of 47.1 months (range 1-141 months). Laparoscopic ventral and incisional hernia repair, based on the Rives-Stoppa technique, is a safe, feasible, and effective alternative to open techniques. More long-term follow-up is still required to further evaluate the true effectiveness of this operation.

Preliminary experience with new bioactive prosthetic material for repair of hernias in infected fields.

Surgisis (Cook Surgical, Bloomington, Ind., USA) is a new four-ply bioactive, prosthetic mesh for hernia repair derived from porcine small-intestinal submucosa. It is a naturally occurring extracellular matrix which is easily absorbed, supports early and abundant new vessel growth, and serves as a template for the constructive remodeling of many tissues. As such, we believe that Surgisis mesh is ideal for use in contaminated or potentially contaminated fields in which ventral, incisional, or inguinal hernia repairs are required. From November 2000 through May 2002, 25 patients (11 male, 14 female) underwent placement of Surgisis mesh for a variety of different hernia repairs. A total of 25 hernia repairs were performed in our patient population. Fourteen procedures (56%) were performed in a potentially contaminated setting (i.e. with incarcerated/strangulated bowel within the hernia or coincident with a laparoscopic cholecystectomy/colectomy). Eleven repairs (44%) were performed in a grossly contaminated field, including one in which an infected polypropylene mesh from a previous inguinal hernia repair was replaced with Surgisis and one in which necrotic bowel was discovered within the hernial sac. Median follow-up was 15 months with a range of 1-20 months. Of the 25 total repairs, there was one wound infection complicated by enterocutaneous fistula in a patient originally operated on for ischemic bowel. The fistula was in a location independent of the Surgisis mesh. There were no mesh-related complications or recurrent hernias in our early postoperative follow-up period. Surgisis mesh appears to be a promising new prosthetic material for hernia repair, especially in contaminated or potentially contaminated fields. Obviously, long-term follow-up is still required.

Monitoring the conformational fluctuations of DNA hairpins using single-pair fluorescence resonance energy transfer.

We present single-pair fluorescence resonance energy transfer (spFRET) observations of individual opening and closing events of surface-immobilized DNA hairpins. Two glass-surface immobilization strategies employing the biotin-streptavidin interaction and a third covalent immobilization strategy involving formation of a disulfide bond to a thiol-derivatized glass surface are described and evaluated. Results from image and time-trace data from surface-immobilized molecules are compared with those from freely diffusing molecules, which are unperturbed by surface interactions. Using a simple two-state model to analyze the open and closed time distributions for immobilized hairpins, we calculate the lifetimes of the two states. For hairpins with a loop size of 40 adenosines and a stem size of either seven or nine bases, the respective closed-state lifetimes are 45 +/- 2.4 and 103 +/- 6.0 ms, while the respective open-state lifetimes are 133 +/- 5.5 and 142 +/- 22 ms. These results show that the open state of the hairpin is favored over the closed state of the hairpin under these conditions, consistent with previous diffusion fluorescence correlation spectroscopy (FCS) experiments on poly(A)-loop hairpins. The measured open-state lifetime is about 30 times longer than the calculated 3 ms open-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS study for hairpins in diffusion with 12-30 thymidines in their loops. As predicted, the closed-state lifetime is dependent on the stem length and is independent of the loop characteristics. Our findings indicate that current models should consider sequence dependence in calculating ssDNA thermostability. The surface immobilization chemistries and other experimental techniques described here should prove useful for studies of single-molecule populations and dynamics.

Laparoscopic-assisted colonoscopic polypectomy: the Texas Endosurgery Institute experience.

PURPOSE: The advent of laparoscopic surgery has altered the manner by which surgical specialties address pathologies of the abdominal cavity. This advance in technology has also changed colorectal surgery. One of the more common procedures of colorectal surgery is segmental resection for polyps that are large, broad based, or inaccessible for colonoscopic removal. We present a technique combining colonoscopy and laparoscopy to remove troublesome polyps without the need for segmental resections. METHODS: From May 1990 to September 1999 laparoscopic-monitored colonic polypectomies were performed in 47 patients, with a total of 60 polyps being removed. After laparoscopic mobilization of the involved segment of the colon, the proximal bowel is cross-clamped and the colonoscope passed to the involved portion of the colon. The polyp is then presented to the colonoscopist by the laparoscopist facilitating removal. The serosal surface is monitored for any indications of transluminal injury, and the area is repaired if needed. All polyps undergo immediate frozen section analysis. If the pathologic evaluation indicates malignancy then a segmental resection may be performed, otherwise the patients are decompressed and fed within a short time before discharge. RESULTS: The polyps were located most commonly in the ascending colon (18 polyps), transverse colon (12 polyps), and cecum (12 polyps). The most common histopathologic diagnosis was tubulovillous adenoma in 28 polyps followed by villous adenoma in 11 polyps. In three cases histopathologic diagnosis revealed malignancy necessitating segmental resection (1 low anterior resection and 2 right hemicolectomies), which were performed laparoscopically. Patients received a liquid diet within 6 hours, were discharged in an average of 21 hours, and returned to full activity, usually within days. The only complication presented in this group of patients was an umbilical port seroma. Virtually all patients (97 percent) behaved as if only a colonoscopy had been performed. Pain at the trocar sites was managed with acetaminophen 600 mg by mouth as needed. CONCLUSION: Laparoscopic-monitored colonoscopic polypectomy allows patients to undergo removal of colonic polyps without a segmental resection. This less invasive procedure yields recovery times similar to that of colonoscopy alone, and the potential complications of a segmental resection are avoided. All polyps are examined by frozen section, and if a malignancy is encountered, a laparoscopic resection can be performed.

Laparoscopic surgery for stage III colon cancer: long-term follow-up.

BACKGROUND: The role of laparoscopic surgery in the management of colorectal cancer is controversial. This study was undertaken to determine the oncological adequacy, in terms of margins of resection, lymph node harvest, and anastomotic and locoregional recurrence of laparoscopic colectomy in patients with stage III (node-positive) colorectal cancer. METHODS: The results of laparoscopic colectomy in 50 consecutive patients with stage III colorectal cancer operated on at a single hospital between 1991 and 1998 were analyzed with respect to postoperative morbidity, mortality, and long-term survival by the Kaplan-Meier method. Methodical patient follow-up was the mainstay of the study. RESULTS: There were 31 men (52%) and 19 women (38%) with a mean age of 67.7 years (range, 40-88). Low anterior resection was performed in 17 cases, abdominal perineal resection in five cases sigmoid colectomy in 10 cases, left hemicolectomy in six cases, right hemicolectomy in seven cases, transverse colectomy in one case, and subtotal colectomy in four cases. Conversion was necessary in three cases (6%). Major complications included one leak, one pelvic abscess, one perineal wound infection, and three anastomotic strictures early in the experience, with none in the past 4 years. One early death occurred due to massive stroke. Median length of stay was 6 days (range, 3-37). Forty-six patients were staged as CII and four as CI colon cancer. The average number of positive nodes was 5.1 (range, 3-58). The margins of resection were adequate in all patients. Follow-up ranged from 3 to 75 months (average, 29.3; median, 24). Overall cancer-related mortality was 34% (17 patients); three patients died of unrelated causes with no detectable cancer. All who died of cancer had distant disease; three of them also had pelvic recurrence. Mean time of death was 21.7 months. There were no anastomotic recurrences or trocar site implants. Overall 3- and 5-year survival was 54.5% and 38.5%, respectively; cancer-adjusted survival was 60.8% and 49.1%. CONCLUSIONS: Based on this study, laparoscopic colectomy in patients with stage III colorectal cancer is oncologically adequate. It results in a long-term outcome comparable to that of traditional open surgery and is associated with low perioperative mortality and morbidity (lower wound infection rate, lower wound recurrences at trocar sites) and a shortened length of stay.

How to prevent port-site metastases in laparoscopic colorectal surgery.

Various reports concerning port-site metastasis after laparoscopic surgery for colorectal cancer have created a new concern regarding the use of this technique for the treatment of this malignancy. The real incidence is not yet known; neither are its prognostic implications. Numerous experimental studies, both in vitro and in vivo, have been published since 1994. These studies have analyzed the possible role of pneumoperitoneum and carbon dioxide (CO(2)) and pathophysiology, as well as the influence of minimally invasive techniques on tumor response and immunity. There are no definitive results yet, but there is enough evidence to presume that the etiology of this new complication might derive from surgical technique. We present our 8-year experience with laparoscopic surgery for colorectal cancer. We also review our technique for preventing port-site implants. At this writing, we have had no port metastasis in our series of 320 colorectal cancer cases with a mean follow-up period of 54 months. The steps we follow as a routine in all cases of laparoscopic colorectal cancer are (a) fixation of trocars to the abdominal wall, (b) avoidance of touching the tumor, (c) high vascular ligation, (d) intraoperative colonoscopy and intraluminal irrigation with 5% iodine povidone, (e) specimen isolation before extraction from the abdominal cavity, and (f) intraperitoneal and trocar-site irrigation with a tumoricide solution.

Laparoscopic ventral and incisional hernia repair.

Incisional hernia repair poses a difficult problem for the general surgeon because of the high incidence of recurrence (50%) and a reported 10% infection rate. Use of a mesh by the anterior approach to replace or reinforce the defect has marginally reduced the recurrence rate, but not the infection rate, especially in obese patients. With the evolution of minimally invasive surgery, we thought that a potential was present to reduce the postoperative stay, lessen pain, and decrease the incidence of both recurrence and infection. From February 1991 through February 1998, a total of 176 patients with complicated umbilical and incisional hernias have been repaired; the follow-up has been from 1 to 84 months. The complication rate was 5.1%, with an infection rate of 1.7% and a 1.1% incidence of recurrence. Seventeen patients had combined procedures, including cholecystectomy, inguinal hernia repair, and antireflux procedures.

Laparoscopic common bile duct exploration by choledochotomy. An effective and efficient method of treatment of choledocholithiasis.

BACKGROUND: Management of cholelithiasis and choledocholithiasis usually requires two separate teams-the gastroenterologist/surgical endoscopist and the laparoscopic surgical team. This requires two separate procedures that potentially increase the overall morbidity and cost. Laparoscopic common bile duct exploration by choledochotomy (LCBDE-C) averts this problem with a single approach. METHODS: In 1990-1991, unsuspected stones found at laparoscopy with intraoperative cholangiogram done routinely underwent postoperative ERCP. Residual stones had been found after ERCP in 16 of 22 preoperative ERCP patients and we began to seek an alternative technique. Laparoscopic common bile duct exploration by choledochotomy has achieved a high rate of success. RESULTS: Technically successful LCBDE-C has been accomplished in 143 of 148 patients (96.6%). Retained bile duct stones have been found on postoperative cholangiogram in three patients (2.0%), all of which have been successfully removed by postoperative ERCP. Thus 140 or 148 patients had their bile duct successfully cleaned by the one-step technique alone (94.6%). CONCLUSIONS: We believe that most laparoscopic surgeons who have acquired the skills of intracorporeal suturing can be successful at laparoscopic common bile duct exploration by choledochotomy. The disadvantage of T-tube presence will likely be eliminated by future developments with intraoperative antegrade sphincterotomy-like procedures, but the ability to see both proximal and distal biliary tree with the choledochotomy in all cases seems to offer more than adequate results at this point in the evolution of the laparoscopic approach to calculus biliary tract disease.

Prospective comparison of open vs. laparoscopic colon surgery for carcinoma. Five-year results.

UNLABELLED: Laparoscopy for colonic diseases began in 1990 and has established a role in benign disease. Early observations and experiences demonstrated feasibility of laparoscopic surgery for a variety of colonic disease processes, but the applicability to colonic carcinoma was unclear. METHODS: In 1990, we began a comparative study of open (OCR) vs. laparoscopic (LCR) approach to colon cancer. The study progressed 65 months, with 224 patients in OCR group and 191 patients in LCR group. Parameters studied are stage, location, length of specimen, number of lymph nodes resected, margins, postoperative course, wound complications, recurrence rates, and immediate and long-term survival. OCR were standardized by one group, and LCR were standardized by a second group. All patients undergoing LCR were given freedom to choose either OCR or LCR, and informed consent was obtained. RESULTS: Equal or greater lymph node retrieval, resections, and distal margins were evident with LCR. Benefits with LCR were shown with shorter hospitalization (5.7 vs. 9.7 days), less blood loss, less wound problems (1 vs. 14), and quicker return of bowel function. Survival, recurrence, and death rates were essentially the same. There were no trocar implants in the LCR group. CONCLUSION: After five years, this study shows that laparoscopy does no harm to the patient, offers comparable oncologic resections, and seems to be patient-friendly, with less pain, quicker return of bowel functions, shortened hospitalization, and quicker return to full activity.

Inflammatory breast cancer.

Inflammatory breast cancer has historically carried a poor prognosis. This has led to the development of multimodal protocols in an attempt to improve survival. Twenty-three women were treated for inflammatory breast cancer at our institution between 1979 and 1992. The mean age at diagnosis was 55.8 years (40 to 77 years). Eighteen women (78%) presented clinically with an erythematous or swollen and tender breast, and 19 (80%) had pathologically demonstrated dermal lymphatic invasion. Five (21.7%) had evidence of distant metastasis at the time of presentation. Treatment consisted of modified radical mastectomy in 65% of patients in combination with preoperative or postoperative chemotherapy. The most common chemotherapeutic regimen was 5-Fluorouracil, Adriamycin, and Cyclophosphamide. Eleven women (48%) also received chest wall irradiation (4,200 to 6,000 cGy). Eleven women had classic multimodality therapy (surgery, chemotherapy, and radiation therapy). Median survival is 23.4 months (6 to 77 months). We concluded that with combination therapy, selected patients can experience long-term survival; however, overall prognosis remains poor, with eventual disease recurrence and death resulting from the disease.

Babesiosis in pregnancy.

Babesiosis is a malaria-like illness due to intraerythrocytic protozoan parasites. To the authors' knowledge, this unusual disease has not previously been described in a pregnant woman. Herein is reported the case of a gravid woman with an intact spleen who developed infection with Babesia microti in the fifth month of gestation. Her illness resolved following supportive care only, and evidence of transmission of disease to the fetus was not found.

Treatment of Crohn's disease with 6-mercaptopurine. A long-term, randomized, double-blind study.

To test the effectiveness of 6-mercaptopurine (6-MP) in the treatment of Crohn's disease, we entered 83 chronically ill patients into a two-year double-blind study comparing 6-MP with placebo. Crossover data showed that improvement occurred in 26 of 39 courses of 6-MP (67%) as compared with three of 39 courses of placebo (8%) (P less than 0.001). Non-crossover data likewise confirmed the superiority of 6-MP. The drug was more effective than placebo in closing fistulas (31 vs 6%) and in permitting discontinuation or reduction of steroid dosage (75 vs. 36%) (P less than 0.001). The onset of response to 6-MP was often delayed, with 32% of patients taking longer than three months to respond, and 19% taking longer than four months. Adverse side effects to 6-MP occurred in 10% of patients and were uniformly reversible. We conclude that 6-MP is an effective and useful agent in the management of Crohn's disease.