D. Alfani

Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

UMAN organs for transplantation are insufficient in quantity, and for every organ transplant undertaken there is a need for an additional five to ten organs. Waiting lists are constantly growing and many patients die before an organ can be found. Researchers continue to experiment with alternatives. One of these is xenotransplantation, which uses animals as donors and which presents an entirely new set of challenges. The pig is presently considered the most likely source of organs for human xenotransplantation because it is easy to breed, has compatibly sized organs, and offers the possibility of genetic manipulation. However, organ transplantation between distantly related species, such as pigs and humans, results in hyperacute rejection (HAR), involving the complement system. It may be possible to avoid rejection reactions by genetically engineering donor animals, so that the recipient’s immune system does not act on the graft. Thus, a number of research teams, including our group, have embarked on programs to produce pigs transgenic for the human regulators of complement activation (RCAs) genes, in the attempt to produce pigs whose organs may be suitable for transplantation into humans. 1‐3 The present study reports on the production of pigs transgenic for the human regulator of complement activation human decay accelerating factor (hDAF) by spermmediated gene transfer (SMGT), a highly efficient and reproducible alternative to microinjection, presently the most widely used system for generating transgenic animals.

Alternative laparoscopic management of perforated peptic ulcers

Surgery—namely, suture closure-is still the treatment of choice for perforated peptic ulcers, despite the proven efficacy of Taylors conservative approach. Such conservative management, however, has been proven less effective in high-risk patients and those with perforations more than 12 h old. Here we suggest alternative laparoscopic treatments for perforated peptic ulcers.We have treated laparoscopically six patients (one F, five M; mean age 57.6 years; range 31–81 years); the mean duration of the operation was 52 min; the median hospital stay was 7 days (6–15 days); H2-blockers, antibiotics, and fluids were administered in the p.o. course; the follow-ups range from 6 to 18 months.On the basis of our experience, the treatment of choice for perforated peptic ulcers is Taylors conservative procedure and laparoscopic drainage of the abdominal cavity when there is mild peritoneal reaction (usually less than 6 h from the onset of perforation). In case of remarkable peritonitis (usually more than 12 h), it is mandatory to add an accurate lavage. When the site of perforation is concealed by the peritoneal inflammation it should not be searched; when visible, it might be obliterated with the round ligament or an omental tissue strand, particularly if larger than 1 cm in diameter.

Immunoadsorption with protein A in humoral rejection of kidney transplants

The presence of alloantibodies may play a role in accelerated or acute humoral rejection. Different therapeutic strategies based on a removal of anti donor antibodies and prevention of their resynthesis have been used in the management of transplant rejection episodes. Immunoadsorption with staphylococcal protein A, a method to selectively remove immunoglobulin G, may represent a new treatment to reverse humoral rejection in kidney transplantation. From 1991 to January 1996, such a method was used in 23 patients in whom an acute humoral rejection developed over a mean period of 14.1 +/- 9.5 days after operation. Twenty-two patients had been transplanted from living donors and one from a cadaveric donor. The ages ranged from 23 to 58 years (mean, 34 +/- 10 years). All transplants were performed according to a negative direct crossmatch. Basic immunosuppression included cyclosporine, steroids, azathioprine, and antilymphocyte globulin or monoclonal antibodies (OKT3). Rejection was diagnosed on the basis of hematochemical tests, Doppler ultrasonography, and kidney biopsy. Only steroid and monoclonal and polyclonal antibody resistant rejections with > 165% positive direct crossmatches against the donor were treated with Protein A immunoabsorption. The procedure used is based on the treatment of 2-3 plasma volumes for the first 2 days and then every other day until a negative crossmatch is obtained, together with improvement in clinical status (mean treatments, 7.3 +/- 4.5 [range, 4-23]; mean duration of treatment, 12.3 +/- 10.2 days [range, 3-44]). From the start of treatment, azathioprine is replaced by cyclophosphamide at a dose of 1-2 mg/kg/day. During treatment, a remarkable fall in immunoglobulin G levels is achieved on the first day, whereas immunoglobulin M titers remain constant, with a slight decrease in serum albumin. Immediately after treatment, a negative crossmatch was found in 22 (95.6%) of 23 patients. In six patients (26%), graft function did not recover, and one patient (4.3%) died. Preliminary results show that immunoabsorption with staphylococcal protein A may be an effective support in the treatment of humoral acute rejection, particularly when it is performed as soon as an early diagnosis of humoral rejection is made. In fact, such treatment has a highly selective adsorption, allows treatment of large volumes of plasma, and can achieve a rapid decrease in the titer of circulating immunoglobulins.

Protection from complement‐mediated injury in livers and kidneys of transgenic mice expressing human complement regulators

Abstract: The major problem in the use of phylogenetically distant donors is a fast, strong reaction called hyperacute rejection. This reaction mediated by complement is directed against the vascular endothelia of the transplanted organ. Complement activation is tightly controlled by several regulatory proteins which inhibit the formation and function of different complement components. To verify the hypothesis that organs expressing such inhibitory factors could be spared from complement‐mediated hyperacute rejection, we have generated mice transgenic for the human complement inhibitor membrane cofactor protein (hMCP) and decay accelerating factor (hDAF). Different levels of hMCP and/or hDAF expression, according to the promoter used, were detected by RNA analysis in the major organs, specifically on the organ vascular endothelia, as revealed by immunohistochemical analysis. The development of an in vivo model of human plasma perfusion allowed the characterization of complement‐mediated damage in control animals and the degree of protection due to the presence of hMCP, hDAF, or both in the organs derived from single or double transgenic mice. In this paper we compare the level of expression of complement regulators with the degree of protection in two major organs: liver and kidney.

Kidneys derived from mice transgenic for human complement blockers are protected in an in vivo model of hyperacute rejection.

PURPOSE The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage. MATERIALS AND METHODS Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 microL./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination. RESULTS Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both hDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage. CONCLUSION A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.

The role of preoperative investigations in predicting difficult laparoscopic cholecystectomies

AbstractBackground: When we began laparoscopic cholecystectomy (LC) we set up a strict preoperative workup in order to assess whether currently available investigations could help predict difficult laparoscopic procedures. Methods: Reported here are the results of a prospective trial carried out in our first 200 consecutive patients, who underwent routine intravenous cholangiography (IVC), abdominal ultrasound scan (US), blood tests—namely, markers of biliary stasis (MBS)—and preoperative endoscopic retrograde cholangiopancreatography (ERCP) in case of clinically suspected common bile duct stones (CBDS). Results: On the basis of our experience we think that the US findings relate to the difficulty of the laparoscopic procedure more closely than the other preoperative investigations, and the association of US and liver chemistry provides an accurate evaluation of biliary stones. Conclusions: In agreement with data emerging from the literature, the preoperative investigations do not seem to be useful in predicting biliary and vascular complications, whose prevention lies in the adoption of correct surgical technique and a low threshold for conversion.

Living kidney transplantation between spouses: results in 100 cases.

Abstract The use of unrelated living donors in kidney transplantation is still controversial but many transplant centres have accepted this procedure. The main argument against this approach is usually an ethical one. Because of this, at our institution we accept biologically unrelated donors only if they have an emotional closeness to the recipient. From January 1983 to October 1993, out of 654 kidney transplantations we performed at our institution, 364 kidney allografts were from living donors. Of these living donors, 245 were first‐degree relatives of the recipient (LRD) while 119 were unrelated (LURD); 100 cases were spouses‐wife to husband in 76 cases and husband to wife in 24 cases Statistical analysis of the results (chisquare) revealed actuarial patient and graft survival rates of 89.8% and 86.8% at 1 year, 82.9% and 72.3% at 5 years and 12.3% and 60.3% at 9 years, respectively. In our series, the result of living donor kidney transplantation in this group were similar to those obtained in the LRD group, while they were significantly better than those from cadaver donors (P = 0.003). In conclusion, cadaver organs given the shortage of kidney transplantation between spouses may be a good alternative and can be performed successfully, providing a “gift of life” for both the patient and the family.