Biomedical subjects
Publications and source records attributed to M Masetti.
The authors present a case of arterial jump graft using a number 9 Goretex prosthesis (FEP ringed vascular graft; W.I. Gore Associates Inc.-Delaware, Flagstaff, AZ) with an excellent outcome 3 years after the transplant. The prosthesis was necessary because of the impossibility of using the donor iliac arterial grafts due to the presence of widespread atherosclerotic damage.
Islet cell transplantation can potentially normalize blood glucose levels and stop the progression of clinical complications, and if the transplant is done early in the course of the disease complications may be prevented. Remarkable progress has been made in recent years and islet cell transplantation has resulted in normalization of metabolic control in several patients with Type 1 diabetes in the absence of hypoglycemia. Only a few patients, however, have achieved insulin independence. Issues relating to islet cell engraftment within the liver, prevention of rejection and recurrent autoimmunity, and identification of alternative immunosuppressive drugs that do not adversely affect islet cell function remain to be solved. Thus far, the need for chronic, generalized immunosuppression to prevent rejection of the islets has limited the indication to those patients who have already received another transplant or to those who simultaneously receive islets and another organ (generally a kidney). Identification of immunointervention protocols that allow for engraftment in the absence of deleterious effects on the islets and prevent rejection and recurrent autoimmunity would make this procedure suitable for all patients, including children who have not yet developed long-term complications of the disease.
During embryonic development, insect vitellins (Vt) are degraded by limited proteolysis to yield a number of lower-molecular weight polypeptides. The aim of the present study was to identify these polypeptides in the embryo and to verify how they relate to Vt polypeptides deposited in the oocyte during vitellogenesis. To this end a panel of poly- and monoclonal antibodies (Pab, Mab) was raised against Vt polypeptides and employed by immunoelectrophoresis and immunoblotting on embryos belonging to different developmental stages. Through this approach three major staining patterns were observed. First, Mab 4 reacts with both polypeptides B1 and E20, suggesting that polypeptide B1 is gradually trimmed to yield polypeptide E20 in late embryos. Second, Mab 12 is specific for polypeptide A3 which is retained unchanged throughout embryogenesis. Third, Pab anti-A2 and Mab 13 show that polypeptide A2 is processed to yield polypeptide E9 through limited proteolysis. In conclusion, the staining patterns reported in this study show that Vt polypeptides in developing embryos of the stick insect Carausius morosus undergo at least two major processing events concerning polypeptides B1 and A2.
Synthesis and secretion of vitellogenin (Vg) polypeptides were studied in egg-laying females of the stick insect Carausius morosus following in vivo exposure to [35S]-methionine and acetyl-N-[3H]-glucosamine. The specificity of radioisotope incorporation was assessed by in vitro inhibition with tunicamycin and carbohydrate extraction with endo-glycosidase H. Vg polypeptides change in molecular weight during synthesis in the fat body and are not further modified upon transfer to the haemolymph or to the oocyte, suggesting that they are already fully glycosylated prior to secretion. Radioactivity in the fat body was initially distributed over cisternae of the rough endoplasmic reticulum and gradually transferred to the Golgi apparatus. Within an hour of exposure, electron-dense granules budding from the trans-Golgi network became preferentially labeled. Radioactivity in the ovarian follicle was restricted to the yolk granules of the cortical ooplasm and to the amorphous material lying within the intercellular channels of the follicular epithelium. This amorphous material was also shown to react positively when tested with a monoclonal antibody raised specifically against a Vg polypeptide.
Vitellins from ovarian follicles and newly laid eggs of the stick insect Carausius morosus were examined by ion exchange chromatography on a HPLC Mono Q column. Under these conditions, vitellins from newly laid eggs resolved as two distinct peaks, referred to as VtA and VtB, that eluted at 8.5 and 12.0 min, respectively. On native gels, both VtA and VtB separated into two different variant forms (VtA' and VtA", VtB' and VtB"). By two-dimensional gel electrophoresis, VtA' and VtA" were shown to contain polypeptides A1, A2 and A3. On the other hand, VtB' and VtB" appeared to comprise polypeptides B1 and B2 and B1, A1, A2, B2 and A3*, respectively. A similar Vt polypeptide composition was also observed by size-exclusion chromatography of vitellins from newly laid eggs. Vitellins from early vitellogenic ovarian follicles resolved into a single chromatographic peak at 7.5 min that coeluted with a major peak from the hemolymph of egg-laying females. Ovarian follicles progressively more advanced in development exhibited a more complex chromatographic profile, consisting of three separate peaks. By two-dimensional gel immunoelectrophoresis, vitellins from ovarian follicles appeared to consist of two closely related, immunologically cross-reacting antigens that gradually shifted apart as ovarian development proceeded to completion. By size-exclusion chromatography, each Vt from ovarian follicles was shown to consist of a unique set of polypeptides different from those listed above. Single ovarian follicles were fractionated into yolk granules and yolk fluid ooplasm and tested by immunoblotting against Mab 12. Under these conditions, VtA variant forms in yolk granules and yolk fluid ooplasm reacted differently. Sections from ovarian follicles in different developmental stages were exposed to Mab 12 and stained with a peroxidase-conjugated, goat anti-mouse antibody. Regardless of the developmental stage attained, staining for peroxidase was restricted to free yolk granules, suggesting that native vitellins in stick insects are structurally modified upon fusion into the yolk fluid ooplasm.
Liver transplantation with preservation of the recipient vena cava (the "piggy-back" technique) has been proposed as an alternative to the traditional method. We performed a randomized study on 39 cirrhotic patients, 20 who underwent the piggy-back technique (group 1) and 19 the traditional method using venovenous bypass (group 2) to evaluate the feasibility and true advantages of the piggy-back technique compared to the traditional method. Two patients were switched to the conventional technique due to the presence of a caudate lobe embracing the vena cava in one patient and a caval lesion in the other. Statistically significant differences between the two groups were only found for the warm ischemia time (48.5 +/- 13 min for piggy-back vs 60 +/- 12 min for the conventional method) and for renal failure (zero cases in group 1 vs four cases in group 2). We therefore believe that liver transplantation with the piggy-back technique can easily be performed in almost all cases, and that only a few, specific situations, such as a very enlarged caudate lobe, do not justify its routine use.
The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.
Undifferentiated (embryonal) sarcoma of the liver is a rare malignant mesenchymal tumor with a poor prognosis. Thirty cases worldwide have been reported over the last 40 years. The absence of specific symptoms, the rapid tumor growth, the normality of the common tumoral markers and the consequential delay in the diagnosis, often allow a significant enlargement of the hepatic mass. Three patients ages 15, 25, and 60 were admitted to our department with such a tumor. In spite of the large dimensions of the neoplasms, all underwent a right hepatectomy without any major surgical complications. Two of them died due to tumor recurrence after 10 and 67 months, respectively. The one case remaining is alive and disease free 62 months postoperatively. The latter one was the only case where a complete tumoral capsule was found in the resected specimen. Surgery is strongly recommended for every case with evidence of a liver-confined tumor even if it is large.
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A retrospective study was carried out of 522 elective liver resections to determine the impact of blood transfusion on the immediate postoperative outcome and on long-term survival. The number of liver resections without transfusion has increased in recent years, as a result of improvement in surgical technique with less blood loss during operation and more careful choice of the timing of transfusion. In resections carried out in the past 5 years, the indication for intraoperative transfusion was restricted and the decision was made jointly by the surgeon and anaesthetist, and in any case only if the haematocrit was below 25 per cent. Of resections carried out in the past 2 years, 59 per cent did not require intraoperative transfusion. Postoperative deaths and complications were related to blood transfusion, particularly in patients with cirrhosis, in whom stepwise logistic regression analysis showed that transfusion was the only factor that correlated significantly with complications. Transfusion also affected the long-term survival of patients operated on for hepatocellular carcinoma and colorectal carcinoma metastases in univariate analysis and was the only factor shown by multivariate analysis to correlate with survival for hepatocellular carcinoma in patients with cirrhosis.
A monoclonal antibody raised against ovarian follicles of the stick insect Carausius morosus reacted with two related polypeptides of 157 and 85 kDa in both the ovary and the hemolymph. In vitro cultured fat body proved capable of releasing the 157-kDa polypeptide into the culture medium and processing it to the lower-molecular-weight polypeptide of 85 kDa. This was further demonstrated by in vitro exposure to [35S]methionine. Under the same culturing conditions, ovarian follicles proved incapable of synthesizing and/or secreting the 85-kDa polypeptide. However, in vivo [35S]methionine-labeled ovarian follicles released both polypeptides when cultured in vitro for up to 24 hr. Vitellogenin polypeptides were labeled in vivo following exposure to [3H]glucosamine, while 157- and 85-kDa polypeptides were labeled only in ovarian follicles exposed in vivo to sodium [35S]sulfate. Under in vitro conditions, the 157-kDa polypeptide could be labeled with sodium [35S]sulfate only if ovarian follicles were cocultured with fat body. No sulfation occurred in fat body or ovarian follicles cultured separately. These experiments suggest that the 157-kDa polypeptide is a fat body-derived polypeptide that is sulfated upon transfer to the ovarian follicle.
Tissue extracts from larval instars of the sheep nasal bot, Oestrus ovis, were resolved by gel electrophoresis under both native and denaturing conditions. Polypeptides resolved under these conditions were tested by immunoblotting against sera of infested sheep. Of all tissues examined in this study, salivary glands proved to be major immunogens in infested sheep. Salivary gland polypeptides were also detected in the washing solution as larval secretory products (LSP). To a minor extent, a few polypeptides from the larval cuticle were also found to be immunogenic, but they did not contribute to LSP. These results were further corroborated by nasal infestation of rabbits that also developed specific antibodies against larval salivary gland polypeptides from Oestrus ovis.
INTRODUCTION: Living donation in adult liver transplantation (LDLTx) is an important resource because of the waiting list growth. We started a living donor program to overcome the shortage of cadaveric sources. PATIENTS: From May 2001 to May 2003, 36 patients underwent LDLTx: 27 received a right lobe, 8 received a left lobe, and 1 received segments II and III. RESULTS: The 1-year actuarial survival rate was 77.7%, with a mean follow-up, in survivors, of 754 +/- 248 days. Eleven of 27 (40.7%) right lobe recipients died. Among left graft recipients, 3 patients died (33%). We undertook retransplantation in 4 cases, because of 2 "small for size" syndrome, 1 late hepatic artery thrombosis, and 1 early portal vein thrombosis. After a period of 797 days, all 36 donors returned to a normal social and working life. Two donors, who underwent right lobe donation, experienced major complications: 1 case of biliary stenosis, treated by stenting, and 1 case of biliary leak from the cut surface of the liver, requiring laparotomy and abscess drainage. Left lobe donors developed no complications. CONCLUSIONS: LDLTx has a learning curve for experienced liver transplantation surgeons. Our last 18 cases showed better survivals than the first 18 (9 deaths vs 5), even if, in the latter group, we transplanted 8 left livers. In our experience, LDLTx of a left liver graft has an increased risk of "small for size syndrome," but patients, both donors and recipients, report improved outcomes.
Induction with thymoglobulin, a potent anti-thymocyte polyclonal antibody, has been recently reported to allow minimization of postoperative immunosuppression in organ transplantation. The relationship with recurrence of hepatitis C virus (HCV) after liver transplantation (LT) has never been investigated. We report herein on the outcome of 22 HCV+ patients receiving thymoglobulin pretreatment and minimal immunosuppression after liver transplantation. Patient survival and acute rejection rates were good, with remarkably low dosages and levels of immunosuppression achieved with thymoglobulin, and without exposing patients to an elevated risk of rejection. A progressive weaning of the primary immunosuppressant was also possible in the majority of patients without complications. The HCV recurrence rate was similar to that reported in the literature, although lower HCV RNA viral loads were obtained with thymoglobulin and a mild histologic course. Although our results need to be validated in large cohort studies, our experience shows that minimization of immunosuppression with thymoglobulin is effective to protect against rejection and demonstrated a positive impact on HCV recurrence that deserves further investigation.
Adult isolated intestinal and multivisceral transplantation is gaining acceptance as the standard treatment for patients with intestinal failure with life-threatening parenteral nutrition-related complications. We report our 4-year experience with intestinal and multivisceral transplantation. We performed 20 isolated small bowel and seven multivisceral ones, including three with liver. The underlying diseases were mainly short bowel syndrome due to intestinal infarction, chronic intestinal pseudo-obstruction, and Gardner syndrome. Indications for transplant were loss of central venous access in 14 patients, recurrent sepsis in eight patients, and major electrolyte and fluid imbalance in five patients. One-year patient actuarial survival rate was 94% for isolated intestinal transplants and 42% for multivisceral recipients (P = .003), while 1-year graft actuarial survival rate was 88.4% for isolated small bowel patients and 42.8% for multivisceral ones (P = .01). The death rate was 18.5%. Our graftectomy rate was 14.8%. Our immunosuppressive protocols were based on induction agents such as alemtuzumab, daclizumab, and antithymocyte globulins. The majority of our complications were bacterial infections, followed by rejections and relaparotomies; most rejection episodes were treated with steroid boluses and tapering. We believe that our results were due to optimal candidate and donor selection, short ischemia time, and use of induction therapy. Multivisceral transplantation is a more complex procedure with less frequent clinical indications than isolated small bowel transplant, but our data concerning multivisceral transplants include only a small number of patients and require further evaluation.
Multivisceral transplants are gaining acceptance worldwide for patients with chronic gastrointestinal failure with or without irreversible total parenteral nutrition (TPN)-related liver failure. We describe our experience with nine multivisceral harvests reporting our in vivo technique. Multivisceral grafts included stomach, duodenum, pancreas, small bowel, and part of large intestine with or without the liver. After a careful evaluation of the liver and the bowel, we isolated the superior mesenteric artery origin. Then we identified the distal part of the graft isolating the middle colic vein and stapling the transverse colon to its left. After esophagus isolation and stapling, we mobilized the graft, starting from the spleen to the pancreaticoduodenal block, near the celiac trunk. After cross-clamping and cold perfusion, we created an aortic patch including the superior mesenteric artery and celiac trunk as a multivisceral harvest without the liver. A total hepatectomy is added for a liver multivisceral graft. We harvested four multivisceral grafts without the liver and five multivisceral grafts with the liver. We performed seven multivisceral transplants on adult recipients, four without the liver and three with the liver, as well as two liver and one isolated small bowel transplants. Postreperfusion hemostasis was always satisfactory with a mean ischemia time of 6.5 hours. Four recipients died: there was one intraoperative death due to disseminated intravascular coagulopathy. Another patient underwent graftectomy 1 day after transplantation due to vascular thrombosis. In conclusion, our in vivo technique allows a shorter ischemia time with a minimal postreperfusion bleeding and reduced production of lymphatic ascites, without jeopardizing organ function.
PURPOSE: We report our experience with intestinal and multivisceral transplantation in Italy. METHODS: We performed 23 adult isolated intestinal transplants and seven multivisceral ones, three with liver, between December 2000 and June 2005. Indications for transplantation were loss of venous access (n = 14), recurrent sepsis (n = 10), and electrolyte-fluid imbalance (n = 6), 14 of whom also presented with total parenteral nutrition (TPN)-related liver dysfunction. Immunosuppression was based on induction agents like daclizumab (followed by tacrolimus and steroids) in the first period; alemtuzumab or thymoglobulin (with tacrolimus) in a second period after 2002. RESULTS: The mean follow-up was 742 +/- 550 days. Three-year patient actuarial survival rate was 88% for intestinal transplants and 42% for multivisceral (P = .015). Three-year graft actuarial survival rate was 73% for intestinal patients and 42.8% for multivisceral (P = .1). Graft loss was mainly due to rejection (57%). Complications were mainly represented by bacterial infections (92% of patients), relaparotomies (82%), and rejections (72%). Full bowel function without any parenteral nutrition or intravenous fluid support was achieved in 60% of recipients with functioning bowel including 95% on a regular diet. One patient underwent abdominal wall transplantation as well. DISCUSSION AND CONCLUSION: Intestinal transplantation has achieved high rates of patient and graft survival with even longer follow-up. Early referral of patients, especially in cases of TPN-liver disease, is mandatory to obtain good outcomes and avoid high mortality rates on the transplant waiting list. Immunosuppressive management remains the key factor to increase the success rate.
Transplantation of pancreatic islets of Langerhans as a therapeutic approach for treatment of type I diabetes offers an alternative to subcutaneous insulin injections. Normalization of blood glucose levels by transplanted islets may prevent the development of diabetes-related complications. Problems related to rejection, recurrence of autoimmunity, and local inflammation upon transplantation of islets into the liver need to be solved before the implementation of islet cell transplantation can be viewed as a justifiable procedure in a large cohort of patients. Islet cell isolation has been quite successful in small animals, but the translation of this approach to nonhuman primates has been less rewarding. One of the main problems encountered in nonhuman primate models is the difficulty of isolating an adequate number of functional islets for transplantation. The aim of the present study was to develop a method for isolating a sufficient number of viable islets from nonhuman primates to allow for reversal of diabetes. By implementing minor modifications in the automated method for human islet isolation we were able to obtain viable, functional islets that responded normally to glucose stimulation in vitro. These islets were also able to reverse diabetes in immunocompromised nude mice, rendered diabetic by streptozotocin. This method of islet cell isolation has enabled us to proceed with protocols of allogeneic islet cell transplantation in preclinical, nonhuman primate models.