The development of new density gradient media for purifying human islets and islet-quality assessments

Successful islet transplantation is dependent on the quality and quantity of islets infused. Islets are purified on density gradients, but procedures currently used have limited capacity for pancreatic digests, islet yield, and viability. We aimed to improve islet purification with a modified gradient medium. Biocoll was diluted in University of Wisconsin solution to create linear density gradients of 1.065 to 1.095 g/mL. Properties of islets purified from 22 human pancreas digests with modified medium were compared with 15 preparations using standard medium. The modification increased the capacity of gradients for pancreatic digests from 20 to 60 mL, islet yield increased from 218,000 to 435,318 per isolation, and viability increased from 65.4% to 92.1%. Islet fractions contained greater than 95% of recovered insulin. Islets showed good physiologic responses to secretagogues and restored normoglycemia in streptozotocin-induced diabetic severe combined immunodeficiency disease mice. The new medium enhances yield, purity, and viability of human islet preparations for clinical islet transplantation.     

Liposome-mediated transfer of vascular endothelial growth factor cDNA augments survival of random-pattern skin flaps in the rat.

Tissue engineering is an application for gene therapy that is in its infancy. We show that simple liposomal-mediated gene transfer could result in a potentially useful biological effect in the field of wound healing. cDNA encoding the 165 amino acid form of vascular endothelial growth factor complexed to commercially available liposomes was injected into rat skin 1 week before raising a random pattern 3 x 10 cm flap. The flap survival was enhanced by 14 percent, and was accomplished without accessing the arterial inflow of the territory. These results were statistically significant (p<0.002) and reproducible. No adverse effects were seen. Histological analysis of the angiogenesis localized much of the new vessel formation to the area around the hair follicles. Polymerase chain reaction amplification of extracted flap tissue confirmed the presence of the transgene.     

Monitoring systemic donor lymphocyte macrochimerism to aid the diagnosis of graft-versus-host disease after liver transplantation

BACKGROUND: The diagnosis of graft-versus-host disease (GvHD) after liver transplantation can be difficult because early symptoms are often nonspecific. In this study, the presence of donor lymphocyte macrochimerism in recipient peripheral blood was examined as a diagnostic aid for GvHD after cadaveric donor liver transplantation. METHODS: Between 1996 and 2002, 33 liver transplant recipients with a clinical suspicion of GvHD (skin rash, diarrhea, pyrexia, pancytopenia, or anemia, without an obvious alternative cause) were investigated for peripheral blood donor lymphocyte macrochimerism. Donor macrochimerism was determined at the time of first clinical presentation by a low-sensitivity polymerase chain reaction (PCR) to detect donor human leukocyte antigen (HLA) alleles using genomic DNA extracted from recipient peripheral blood. Where donor HLA alleles were detected, the percentage of donor T cells was quantified by two-color flow cytometric analysis using antibodies specific for mismatched donor and recipient HLA alleles. The relationship between the presence or absence of donor lymphocyte macrochimerism and final diagnoses based on clinical and histological criteria was examined. RESULTS: Seven of the 33 patients were PCR positive for donor HLA alleles. All had macrochimerism, with donor T lymphocyte levels ranging from 4% to 50% of circulating lymphocytes. All seven patients had normal liver function tests, skin rash, and diagnosis of GvHD histologically confirmed by skin or gut biopsies. Twenty-six patients were PCR negative, and, in 23, an alternative diagnosis was eventually established. The remaining three patients made a rapid and spontaneous recovery with no further symptoms suggestive of GvHD. CONCLUSIONS: Donor lymphocyte macrochimerism was present in all patients in whom the diagnosis of GvHD was confirmed. In patients with symptoms consistent with GvHD and a negative PCR for donor HLA, an alternative diagnosis was eventually established or the patients recovered spontaneously. Detection of donor HLA alleles in recipient peripheral blood by PCR is a useful diagnostic tool for GvHD after liver transplantation.     

Intrathecal anesthesia and recovery from radical prostatectomy: a prospective, randomized, controlled trial

BACKGROUND: Previous studies suggest that intraoperative anesthetic care may influence postoperative pain and recovery from surgery. The authors tested the hypothesis that the addition of intrathecal analgesia to general anesthesia would improve long-term functional status and decrease pain in patients undergoing radical retropubic prostatectomy. METHODS: One hundred patients received either general anesthesia supplemented with intravenous fentanyl or general anesthesia preceded by intrathecal administration of bupivacaine (15 mg), clonidine (75 microg), and morphine (0.2 mg). Patients and providers were masked to treatment assignment. All patients received multimodal pain management postoperatively. Primary outcomes included pain and functional status over the first 12 postoperative weeks. RESULTS: Patients receiving intrathecal analgesia required more intravenous fluids and vasopressors intraoperatively. Pain was well controlled throughout the study (mean numerical pain scores < 3 in both groups at all times studied). Intrathecal analgesia decreased pain and supplemental intravenous morphine use over the first postoperative day but increased the frequency of pruritus. Pain and functional status after discharge from the hospital did not differ between groups. Intrathecal analgesia significantly decreased the duration of hospital stay (from 2.8 +/- 2.0 to 2.1 +/- 0.5 days; P < 0.01) as a result of five patients in the control group who stayed in the hospital more than 3 days. CONCLUSIONS: The benefits of improved immediate analgesia and decreased morphine requirements resulting from intrathecal analgesia must be weighed against factors such as pruritus, increased intraoperative requirement for fluids and vasopressors, and resources needed to implement this modality. Further studies are needed to determine the significance of the decrease in duration of hospital stay.     

Bradykinin antagonists have no analgesic effect on incisional pain

Bradykinin, an endogenous nonapeptide and an important mediator of inflammation, is also implicated in the initiation and maintenance of pain. Both des-Arg(8), Leu(8)-bradykinin (dALBK) and HOE-140, the prototypic bradykinin B1 and B2 receptor antagonists, respectively, have been shown to reduce pain behaviors and inflammation in animal models of persistent nociception. We studied them for activity against incision-induced pain behaviors in a rat model for postoperative pain. A 1-cm plantar incision was made in the hind paw of halothane-anesthetized rats and closed with 5-0 nylon. Withdrawal responses to punctate and nonpunctate mechanical stimuli were tested with von Frey filaments and a plastic disk attached to a von Frey filament, respectively. Withdrawal latency to radiant heat was also tested. Rats were tested 1 day before the incision, 1 h after the incision, and 0.5, 1, 1.5, and 2.5 h after the injection of the drug. They were then retested at the same times before and after the injection of the drug on each of the first 2 postoperative days. The rats received the saline vehicle dALBK (0.1, 0.3, 1.0, or 3.0 mg/kg) or HOE-140 (0.1, 0.3, 1.0, or 3.0 mg/kg) IV. Another group of rats had the drug injected 1 h before incision and tested as above. Statistical significance (P < 0.05) was determined with Kruskal-Wallis test and a two-way analysis of variance. None of the doses of either dALBK or HOE-140 affected the responses to punctate or blunt mechanical stimulation or heat, either as a pretreatment or as a posttreatment. These data support the unique mechanisms for incision-induced pain relative to inflammation-related pain. Although inflammation may represent a component of incisional pain, the etiology of inflammation and its role seem different than in other models.     

Late incidence and determinants of stroke after aortic and mitral valve replacement

Background

Stroke is a devastating complication in patients with prosthetic valves, but characterization of its late occurrence from a large cohort is lacking.

Methods

Three thousand one hundred eighty-nine adult patients who underwent a total of 3,576 operations for left-heart valve replacement were managed with contemporary anticoagulation guidelines and prospectively followed in a dedicated clinic. Total follow-up was 20,096 patient years. Bootstrapped survival analysis was used to determine the impact of patient and valve related factors on the incidence of stroke.

Results

Most strokes were embolic. Linearized embolic stroke rates were 1.3% ± 0.2% per year for aortic bioprostheses, 1.4% ± 0.2% per year for aortic mechanical valves, 1.3% ± 0.3% per year for mitral bioprostheses, and 2.3% ± 0.4% per year for mitral mechanical valves (p = 0.002, vs other implant types). Age more than 75 years, female gender, and smoking were independent risk factors after aortic and mitral valve replacement. Atrial fibrillation, coronary disease, and tilting-disc mechanical prostheses were independent predictors of embolic stroke after aortic valve replacement. Preoperative left ventricular (LV) dysfunction was an independent risk factor in patients with mitral prostheses. Primary operative indication, diabetes, redo status, or the presence of two prosthetic valves were not associated with an increased hazard. The addition of acetyl salicylic or dipyridamole to warfarin anticoagulation did not significantly lower embolic stroke risk in patients with mechanical prostheses.

Conclusions

Approximately 20% of patients with valve prostheses have an embolic stroke by 15 years after valve replacement. Some risk factors such as the avoidance of smoking, mitral mechanical prostheses, aortic tilting-disc valves, and proceeding to mitral surgery before LV dysfunction occurs are potentially modifiable.     

The open peritoneal cavity: etiology correlates with the likelihood of fascial closure

The use of laparostomy in damage control surgery and uncontrolled intra-abdominal infection has been well described. We examined 71 patients who required laparostomy to see if trends in management and outcome could be identified based on the underlying disease state. The underlying etiology included gastrointestinal sepsis (n = 25), pancreatitis (n = 21), or trauma (n = 25). Pancreatitis patients required more operations per patient (P < 0.05). The likelihood and type of closure (fascial, mesh, or none) was related to the underlying etiology: trauma patients were more likely to have fascial closure (P < 0.02), patients with GI sepsis were more likely to require mesh closure, and pancreatitis patients were more likely to have no formal closure (P < 0.02). Only 29 per cent of patients achieved definitive fascial closure. Mortality in trauma patients was 20 per cent, 36 per cent for GI sepsis, and 43 per cent in patients with pancreatitis. Complications of laparostomy included enterocutaneous fistula (16.9%) and abscess formation (7%). Though the use of laparostomy has become more prevalent, it is still associated with significant hospital stay, morbidity, and mortality. In our study, the number of operations and likelihood of abdominal closure appears to correlate with the etiology of the underlying disease requiring use of laparostomy.     

Iliac compression syndrome and recanalization of femoropopliteal and iliac venous thrombosis: a prospective study with magnetic resonance venography

OBJECTIVES: Poor iliac vein recanalization has been associated with compression of the left common iliac vein by the right common iliac artery (RCIA/LCIV compression); however, this finding has been difficult to confirm. In a baseline study, RCIA/LCIV compression was detected with magnetic resonance imaging in patients with deep venous thrombosis. We compared recanalization of left femoropopliteal and iliac thrombosis with and without RCIA/LCIV compression. METHODS: This was a prospective blinded study carried out in a 1355-bed university hospital. Thirty-one patients were recruited from consecutive cohorts of patients with iliofemoral and femoropopliteal DVT who underwent direct thrombus magnetic resonance imaging, venous enhanced peak arterial magnetic resonance venography, and magnetic resonance arteriography as part of the baseline study relating RCIA/LCIV compression to extent of thrombosis. Magnetic resonance venography was performed 6 weeks, 6 months, and 1 year after diagnosis of deep venous thrombosis. Femoropopliteal and iliac venous segments that were occluded at diagnosis were classified as occluded, partially occluded, or patent on follow-up scans. RESULTS: At 6-week follow-up, recanalization of all segments was incomplete. At both 6-month and 1-year follow-up, recanalization of left iliac segments associated with RCIA/LCIV compression was poorer compared with recanalization of left iliac segments not associated with compression (6 of 6 occluded vs 1 of 6 occluded and 1 of 6 partially occluded at 6 months, P =.015; 6 of 6 occluded vs 5 of 5 patent at 1 year, P = .002). This was due to complete failure of recanalization of left common iliac veins associated with RCIA/LCIV compression in 6 of 6 cases. All other iliac and femoropopliteal segments including left external iliac veins associated with RCIA/LCIV compression had high rates of recanalization at both 6 months and 1 year. CONCLUSION: RCIA/LCIV compression is associated with persistent occlusion of the left common iliac vein. The recanalization rate for all other femoropopliteal and iliac segments was high.     

Donor Kidney Exchanges

Kidney transplantation from live donors achieves an excellent outcome regardless of human leukocyte antigen (HLA) mismatch. This development has expanded the opportunity of kidney transplantation from unrelated live donors. Nevertheless, the hazard of hyperacute rejection has usually precluded the transplantation of a kidney from a live donor to a potential recipient who is incompatible by ABO blood type or HLA antibody crossmatch reactivity. Region 1 of the United Network for Organ Sharing (UNOS) has devised an alternative system of kidney transplantation that would enable either a simultaneous exchange between live donors (a paired exchange), or a live donor/deceased donor exchange to incompatible recipients who are waiting on the list (a live donor/list exchange). This Regional system of exchange has derived the benefit of live donation, avoided the risk of ABO or crossmatch incompatibility, and yielded an additional donor source for patients awaiting a deceased donor kidney. Despite the initial disadvantage to the list of patients awaiting an O blood type kidney, as every paired exchange transplant removes a patient from the waiting list, it also avoids the incompatible recipient from eventually having to go on the list. Thus, this approach also increases access to deceased donor kidneys for the remaining candidates on the list.