[Transperineal sonography in anorectal disorders]. / Transperineale Sonographie bei rektoanalen Erkrankungen.

AIM: Magnetic resonance imaging, computed tomography, endorectal and endoanal sonography are used for imaging of inflammatory and neoplastic conditions of the lower rectum, the anus and the perineum. These methods, however, have their limitations regarding accuracy, cost-effectiveness, and availability in the acute setting. Pain may be a limiting factor when introducing a probe into the anus. Percutaneous transperineal sonography is an acknowledged method for diagnosing anorectal malformations in children and infants and for diagnosis of prostatic disease. Until today, only limited reports regarding the value of transperineal sonography for evaluation of diseases of the lower rectum, the anus and the perianal region in adults are available. PATIENTS AND METHODS: Between 1997 and 2000 a total of 44 patients underwent transperineal sonography including B-mode and colour Doppler sonography for imaging anorectal structures using 3.5 MHz sector arrays and 7 MHz linear arrays. The lesions were also investigated using colour Doppler imaging. During examination the patient is positioned on his side. Orientation landmarks are the pubic symphysis and the prostate gland in men and the vagina in women. RESULTS: The spectrum of diseases of the current study population (44) included perianal fistulas (10), pararectal abscesses (7), fistulas with abscess (7), perianal inflammation without abscess (1), rectal (6) and anal carcinomas (3) and metastasis of a leiomyosarcoma (1). In 10 cases digital examination of the anus and rectum was not possible because of intense pain. In 34 patients (85 %) sonographic findings were confirmed by intraoperative diagnosis. CONCLUSION: Transperineal sonography proved to be an inexpensive, easily available diagnostic tool that may help in detecting a variety of pathological conditions of the lower rectum, the anus, and the perianal region.

Improved technique for infrahepatic vena cava reconstruction in guinea pig-to-rat liver transplantation.

Guinea pig-to-rat orthotopic liver transplantation is associated with serious technical problems contributing to impaired graft perfusion and primary graft failure. In order to shorten the procurement procedure and thereby minimize liver damage before flushing, a simplified technique for infrahepatic caval reconstruction was developed. Dissection of the infrahepatic vena cava (IHVC) from adrenal glands and renal and lumbar veins represents the most difficult and time-consuming part of the donor operation, which is often not well tolerated by the animal; we avoided this step by using an isogeneic vena cava interposition graft (VCIG) following in situ perfusion. This graft is connected with the IHVC transsected just below the liver with a cuff technique. Donor operations lasted 15 to 20 minutes with the new technique (n = 7) compared to 52 to 76 minutes with conventional technique (n = 7). Reduced operating time was associated with markedly improved graft perfusion and significantly better graft survival. This modification of the donor procedure for the guinea pig-to-rat liver xenograft using a VCIG significantly reduces operating time and improves reperfusion and recipient survival.

Transient complement inhibition plus T-cell immunosuppression induces long-term survival of mouse-to-rat cardiac xenografts.

BACKGROUND: The use of anti-B-cell and T-cell immunosuppressive agents leads to only a few weeks' survival of mouse-to-rat cardiac xenografts. METHODS: BALB/c cardiac xenografts were transplanted to Lewis rats treated with cyclosporine (CsA) and/or cobra venom factor (CVF). RESULTS: CsA alone did not prolong xenograft survival (2.2+/-0.4 days), whereas CVF alone led to minimal prolongation of survival (5.6+/-0.8 days) as compared with nontreated recipients (2.4+/-0.5 days). The combination of CsA plus CVF, the latter given for either 2 days or 11 days, resulted in long-term survival of 14/16 hearts (> 100 days). Production of IgM elicited xenoreactive antibodies (EXA) peaked on day 4 after transplantation and decreased thereafter. Production of IgG EXA occurred only in the control group, whereas, in the CsA/CVF-treated group, IgG EXA were totally suppressed. Long-term surviving grafts showed (i) excellent preservation of morphology and minimal leukocyte infiltration, (ii) deposition of IgM, IgG and weak C3 deposition on the graft endothelium, (iii) low level infiltration by rat macrophages, (iv) replacement of mouse dendritic cells by class II+ rat macrophages, and (v) expression within endothelial and smooth muscle cells, macrophages, and myocytes of HO-1, a "protective gene" not seen in the rejected hearts. CONCLUSIONS: Our present findings suggest that long-term mouse-to-rat cardiac xenograft survival is induced by temporary suppression of C activation and sustained T-cell suppression leading to inhibition of IgG EXA production. Florid expression of a protective gene (HO-1) may contribute to survival.

2-Chlorodeoxyadenosine (cladribine) in combination with low-dose cyclosporin prevents rejection after allogeneic heart and liver transplantation in the rat.

The purine analogue 2-chlorodeoxyadenosine (2-CDA) has been shown to possess synergistic immunosuppressive properties when given together with cyclosporin (CSA) in a rat small bowel transplant model. The present study investigated the immunosuppressive potency of 2-CDA alone and in combination after liver or heart transplantation in a fully allogeneic rat model with 5 animals in each group. Immunosuppression was provided with CSA 10 mg/kg body weight (BW)/day orally or 2-CDA 0.1 mg/kg/BW day intravenously or both compounds together in the dosages mentioned. Animals were sacrificed on day 10 following transplantation, and graft histology was assessed. In addition, cardiac graft function was evaluated by palpation immediately prior to sacrificing the animal. CSA given alone was able to mitigate but not prevent rejection. 2-CDA alone did not exhibit any detectable immunosuppressive effect. When CSA was combined with 2-CDA, no rejection was seen in 80% of the liver allografts and in 60% of heart allografts, and only mild rejection was observed in the remaining animals. All hearts of the combined treatment group, however, beat strongly. From these findings it is concluded that 2-CDA alone has no, but together with CSA a strong immunosuppressive effect in preventing solid organ allograft rejection.

[A new method of orthotopic liver transplantation from the guinea pig to the rat]. / Eine neue Technik der orthotopen Lebertransplantation vom Meerschweinchen auf die Ratte.

Guinea pig to rat orthotopic liver transplantation is associated with serious technical problems contributing to impaired graft reperfusion and a high incidence of primary non function. In order to reduce the operation time and thereby organ damage during procurement a simplified technique for reconstruction of the infrahepatic vena cava was developed and is described in detail. Reduced operation time was associated with markedly improved lobular graft perfusion and significantly better graft survival. We suggest a modification of the donor operation for the guinea pig to rat xenograft liver model for the sake of reducing non immunological factors in this difficult setting.

At what stage does pancreas allograft rejection become irreversible?: an experimental study.

BACKGROUND: It is commonly believed that abnormal blood glucose levels indicate irreversible rejection. We were interested in determining the stage at which rejection remains reversible. METHODS: A total of 54 Lewis rats were rendered diabetic with 55 mg/kg streptozocin and were then given a pancreas transplant from Brown Norway donors. Pancreatic juice was collected in a subcutaneous reservoir. All recipients received 15 mg/kg cyclosporine A (CsA) for 5 days. CsA was then discontinued for 2 days (n=7, group 1), 4 days (n=7, group 2), 6 days (n=9, group 3), 8 days (n=9, group 4), 9 days (n=11, group 5), and 10 days (n=11, group 6). Two animals of each group were euthanized at the end of the immunosuppressive-free interval, for histological assessment of the grade of rejection (G0, GI, GII, GIII). Rejection was treated with methylprednisolone (7 mg/kg body weight) and CsA (15 mg/kg body weight). The volume of pancreatic juice, together with juice cytology (C0, CI, CII) and blood glucose levels, was assessed daily. RESULTS: Blood glucose remained normal throughout the observation period in animals with GI and GII rejection. The numbers of animals that became diabetic were as follows: 5 of 9 (group 4), 7 of 11 (group 5), and 8 of 11 (group 6). Decreased amounts of pancreatic juice were observed in all animals, except those in group 1. The histology returned to normal after anti-rejection therapy in four animals (57%) of group 1, in two animals (28%) of group 2, and in one animal (11%) of groups 3 and 4, respectively. Although there was no animal in groups 5 and 6 with normal graft histology after treatment, there were still four (36%) and three (27%) animals, respectively, that were normoglycemic and that had pancreatic grafts with well-preserved islets. CONCLUSIONS: From these data, we conclude that even GIII rejection with severe endothelialitis and isleitis can be reversed. Therefore, we suggest that a trial of enhanced immunosuppression is justified in patients with advanced pancreas allograft rejection.

Accommodation of vascularized xenografts: expression of "protective genes" by donor endothelial cells in a host Th2 cytokine environment.

Organ xenografts under certain circumstances survive in the presence of anti-graft antibodies and complement, a situation referred to as "accommodation." We find that the endothelial cells (ECs) in hamster hearts that accommodate themselves in rats express genes, such as A20 and bcl-2, that in vitro protect ECs from apoptosis and prevent upregulation in those cells of proinflammatory genes such as cytokines, procoagulant and adhesion molecules. Hearts that are rejected do not express these genes. In addition, vessels of rejected hearts show florid transplant arteriosclerosis whereas those of accommodated hearts do not. Accommodated xenografts have an ongoing T helper cell type 2 (Th2) cytokine immune response, whereas the rejected grafts have a Th1 response. We propose a model for factors that contribute to the survival of xenografts and the avoidance of transplant arteriosclerosis.

Apyrase administration prolongs discordant xenograft survival.

Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases. Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.

Immunomodulatory effects of the alkaloid sinomenine in the high responder ACI-to-Lewis cardiac allograft model.

Extracts of the plant Sinomenium acutum have been used safely since ancient times in Chinese medicine for treatment of rheumatic diseases, and the purified alkaloid, sinomenine, was recently shown to have anti-inflammatory and antirheumatic effects. This study describes the effects of sinomenine in the high responder ACI-->Lewis cardiac transplant model in which allograft rejection occurred at 5 days posttransplant. Treatment with sinomenine (15-30 mg/kg/day i.p.) or a subtherapeutic dose of cyclosporine (CsA, 1.5 mg/kg/day, i.m.) prolonged allograft survival only marginally (mean survival of 5.4 and 7.8 days, respectively). In contrast, the combination of sinomenine and CsA had a statistically significant synergistic effect, with a mean survival of 42.2 days (P < 0.001). Allografts harvested at day 5 from recipients treated with either sinomenine or CsA showed dense mononuclear cell infiltrates with widespread subepicardial infarcts, edema, and microvascular platelet and fibrin deposition. Immunohistologic analysis showed that intragraft leukocytes consisted of >75% macrophages with approximately 10-20% T cells and <5% B or NK cells. Mononuclear cell activation was shown by expression of IL-2R (CD25, 10-20%) and labeling for IL-2 (approximately 10%), and IFN-gamma (10-20%), as well as TNF-alpha (>50%) and iNOS (>50%), but only low levels of IL-4 or IL-10 (<5%). Intragraft endothelial cells were activated, as shown by upregulation of MHC class II antigen and ICAM-1 (CD54) compared with only basal levels in normal donors hearts. Combined sinomenine/CsA therapy significantly enhanced graft morphology, resulting in only mild mononuclear cell infiltration, and an absence of infarcts, platelets, or fibrin deposition. Though residual intragraft mononuclear cells at day 5, as in control grafts, consisted primarily of macrophages plus small numbers of IL-2R+ T cells, these cells lacked expression of IL-2, had only low levels of IFN-gamma, but showed dense labeling for IL-4 and IL-10. In addition, TNF-alpha and iNOS were reduced to basal levels and no endothelial cell activation was observed, despite high titers of endothelium-bound IgM, IgG, and C3. Mitogen-induced in vitro proliferation of rat thymocytes was also more effectively decreased by the sinomenine/CsA combination than by either agent alone. These studies demonstrate the therapeutic value of sinomenine in transplantation, and indicate that this agent has novel and interesting antimacrophage, T cell, and endothelial effects that warrant further evaluation.

T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection.

Rejection of guinea pig cardiac grafts in rats depleted of complement takes place in 3-4 days and involves progressive mononuclear cell infiltration and cytokine expression, fibrin and antibody deposition, and endothelial cell up-regulation of adhesion and procoagulant molecules, a process termed delayed xenograft rejection (DXR). The relative contribution of each effector mechanism and the role of T cells in this complex process are unknown, although small numbers of interleukin (IL) 2 receptor-positive T cells are present at the time of rejection. We investigated the importance of T cells in DXR by comparing discordant xenograft responses of nude rats, which lack T cell receptor (TCR)-alpha/beta+ cells, with those of normal Lewis rats. Nude or Lewis rats receiving guinea pig cardiac grafts were assigned to one of three groups: no therapy, daily administration of cobra venom factor (CVF), or splenectomy plus daily CVF. All untreated rats rejected their xenografts within 10-15 min, whereas grafts in complement-depleted recipients survived a further 3-4 days; splenectomy had no significant additional effect upon graft survival. Immunohistologic analysis in CVF-treated nude recipients with or without splenectomy showed: (1) considerable leukocyte infiltration of xenografts (mean +/- SD, 76+/-14 and 71+/-16 leukocytes/field, respectively, at 72 hr, compared with 68+/-17 in Lewis rats), consisting largely of macrophages (>75% of total leukocytes) plus small numbers of natural killer cells (10-20%) with no detectable B or T cells (TCR-alpha/beta or TCR-gamma/delta); (2) at least 10-fold lower levels of intragraft IgM or IgG deposition than in corresponding Lewis recipients; and (3) considerable cytokine expression by intragraft macrophages (IL-12, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, IL-1beta, IL-6, IL-7, IL-12) and natural killer cells (interferon-gamma), as well as up-regulation of tissue factor expression and dense fibrin deposition. Analysis of recipient sera of both control and nude rats by ELISA, for the binding of IgG or IgM to guinea pig platelets, showed a rapid rise after transplantation in the titers of IgM and IgG antibodies, which was abrogated by prior splenectomy; i.e., data from splenectomized xenograft recipients reflect the presence of only basal levels of IgM and IgG. Thus, our data in nude rats show rejection times and intragraft features of DXR comparable to those in immunocompetent Lewis recipients, despite a lack of detectable host T cells, and, in the case of splenectomized rats, only about one tenth of normal xenoreactive antibody levels. Our data document a new model in which to analyze the immunopathogenesis of DXR.