Differential expression of tissue factor (TF) in calcineurin inhibitor-induced nephrotoxicity and rejection--implications for development of a possible diagnostic marker.

Deposition of fibrin in the form of fibrinoid necrosis is a common feature of severe acute renal allograft rejection. The role of the coagulation system and its initiator tissue factor (TF) during this process is, however, still poorly understood. In this study, we analyzed the expression of TF in 88 renal transplants afflicted with different forms of rejection and calcineurin inhibitor-induced nephrotoxicity, to see whether there was differential expression of this protein. TF immunoreactivity was evaluated semiquantitatively in six different renal structures: the podocytes, Bowman epithelium, the endothelium of the glomeruli, the brush border of tubular cells, the thin ascending loop of Henle, and small arteries/arterioles. The TF expression of normal renal tissue (n=6) was restricted to the glomerular podocytes and Bowman epithelium, and to some extent the ascending loop of Henle. Renal allografts undergoing acute rejection (AR) of grades I-III, (n=13, n=17 and n=12, respectively) did not show any altered TF expression in the glomeruli or vascular endothelium. In the ascending loop of Henle, a reduced expression could be seen (ARI, p=0.015; and ARII, p=0.043). TF staining of the brush border of renal transplants undergoing acute cyclosporin A (CsA) nephrotoxicity (n=18) was significantly higher than in normal kidneys (p=0.0003), as well as in transplants undergoing various degrees of acute rejection (ARI, p=0.027; ARII, p=0.0012; and ARIII, p=0.0001). Tubular brush border-expressed TF was also evident in 10 of 15 allografts suffering from chronic CsA nephrotoxicity, compared to 4 out of 13 cases with chronic allograft vasculopathy (CAV), but the increase was not statistically significant relative to normal kidneys. The majority of the grafts afflicted with either of the two chronic conditions displayed a TF-positive arterial endothelium (CAV, p=0.0034; and chronic CsA nephrotoxicity, p=0.0026) relative to controls. In conclusion, these results indicate that vascular TF expression is not altered during acute rejection, but may be of importance in chronic allograft nephropathy. Furthermore, TF immunoreactivity in the tubular brush border may be specific to acute CsA nephrotoxicity and might be used as a biomarker for this condition. Further studies are required to evaluate the possible role of brush border-expressed TF in the pathogenesis of CsA nephrotoxicity.

Does mesh implantation affect the spermatic cord structures after inguinal hernia surgery? An experimental study in rats.

BACKGROUND: Inguinal hernia repair is the most common operation in general surgery. Prosthetic reinforcement of the inguinal area with polypropylene mesh has increased dramatically in the last decade. The aim of this study was to evaluate how different types of mesh affect the spermatic cord structures. METHODS: Thirty rats were divided into three groups. The spermatic cord was dissected free and a conventional suture repair was performed in group I, an operation mimicking the Lichtenstein operation with a heavyweight polypropylene mesh in group II and the same operation using large pore, lightweighted polypropylene/polyglactin composite mesh in group III. A vasography was performed after 90 days. The cross-sectional area of the vas deferens and s-testosterone from the spermatic vein were measured using the contralateral side as control. Light microscopy of the inguinal canal was performed and inflammation and fibrosis were graded. RESULTS: Vasography revealed patent vas deferens in all animals. In group III, there was a lower s-testosterone in the spermatic vein and a reduced cross-sectional area of the vas deferens on the operated compared to the control side. However, there was no difference in the other groups and there was no significant difference in s-testosterone levels between the groups. There was significantly more inflammation and fibrosis after mesh repair compared to suture repair, but there was no difference between the two mesh groups. Unexpectedly, polyglactin fibres were still seen in specimens in group III after 90 days. CONCLUSION: In conclusion, the only effect on the spermatic cord structures in a rat model is seen as an impaired s-testosterone production and a reduced cross-sectional area of the vas deferens after use of a low-weight composite mesh compared to the control side. No difference in inflammation or fibrosis was found between heavyweight polypropylene mesh and low-weight composite mesh.

Rectal endosonography can distinguish benign rectal lesions from invasive early rectal cancers.

OBJECTIVE: To determine whether an experienced ultrasound examiner, using good ultrasound equipment with high multifrequency probes, can discriminate between a high grade or low grade dysplastic adenoma (pT0) and very early invasive rectal cancers (pT1). SUBJECTS AND METHODS: Sixty consecutive patients with clinically possibly pT0 or pT1 rectal tumours referred for transanal local excision underwent endorectal ultrasound examination. Lesions where the endorectal ultrasound image showed the mucosal layer to be expanded but the submucosal layer to be intact (uT0) were considered to represent a low grade or high grade dysplasia adenoma (pT0). An irregularity or disruption of the submucosal layer (uT1) was considered to characterize early invasive rectal cancers (pT1). The ultrasound staging was compared with the histological staging made on the basis of the diagnoses in the excised specimens. RESULTS: The histopathological diagnoses were: invasive rectal cancer (n = 18, 10 pT1, 4 pT2, 4 pT3 cancers); high grade dysplastic adenoma (n = 21); low grade dysplastic adenoma (n = 18); non adenomatous benign lesions (n = 3). Endorectal ultrasound incorrectly classified two of the invasive cancers (both pT1 tumours) as noninvasive lesions. Five of 42 pT0 tumours were overstaged as uT1 tumours. Overstaging was more common in patients who had undergone a previous excision and in tumours with peritumoral inflammation and desmoplastic reaction. The sensitivity of endorectal ultrasound with regard to invasive cancer was 89% (16/18), specificity 88% (37/42), positive predictive value 76% (16/21), negative predictive value 95% (37/39), and accuracy 88% (53/60). Among pT0 and pT1 tumours, the corresponding figures were 80% (8/10), 88% (37/42), 62% (8/13), 95% (37/39), and 87% (45/52). CONCLUSION: Endorectal ultrasound can distinguish between noninvasive lesions and invasive rectal cancers clinically of stage pT0 or pT1.

Factor V R506Q mutation (activated protein C resistance) is an additional risk factor for early renal graft loss associated with acute vascular rejection.

BACKGROUND: The factor V R506Q mutation (FV R506Q, FV:Q506, or FV Leiden) resulting in activated protein C (APC) resistance is the most common inherited risk factor for venous thrombosis, including in renal transplant recipients. We investigated a possible association between the FV mutation and early renal graft loss, and the prevalence of macro- and microvascular thrombosis, endothelialitis, and fibrinoid vascular necrosis by FV genotype. METHOD: One hundred and nine renal allograft recipients were genotyped for FV mutation. A vascular rejection subgroup of patients (n=29) had experienced at least one episode of vascular rejection, or graft thrombosis. A second group of patients (n=80) had experienced no acute rejection and retained a well-functioning graft. RESULTS: The prevalence of APC resistance was numerically but not statistically significantly higher in the vascular rejection group (17.2%) compared with the group without rejection episodes (7.5%) (P=0.16). There was a significant association between the presence or absence of FV mutation and graft survival, with a 55.6% 1-year graft survival rate versus a 76.4% rate, respectively (P=0.02). The prevalence of vascular rejection, as evidenced by endothelialitis or fibrinoid vascular necrosis, was significantly associated with APC resistance but macro- or microvascular thrombosis were not. CONCLUSION: Renal transplant recipients who are carriers of the FV:Q506 allele have an increased risk of early graft loss. Vascular rejection changes including endothelialitis and fibrinoid vascular necrosis were more common in this group, and therefore an association between the hypercoagulable state, which entails an up-regulation of the mitogenic and proinflammatory enzyme thrombin, and the immunological challenge to the endothelium may be the cause of inferior prognosis in these patients.

Malononitrilamides and tacrolimus additively prevent acute rejection in rat cardiac allografts.

The novel immunosuppressive agents malononitrilamides (MNA) 279 and 715 are derivatives of A77 1726, the primary metabolite of leflunomide. The effects of these agents have been previously demonstrated in rat skin and cardiac allo- and xenotransplant models. The aim of this study was to evaluate the combination of MNA and tacrolimus in a high-responder rat cardiac allotransplant model. Graft survival was evaluated following 10 days of post-transplant oral therapy of MNA or tacrolimus alone and the drugs combined in PVG recipients of DA grafts. An iso-effect curve of single and combined drugs was constructed. Histological changes in grafts were evaluated at 10 days. MNA (20 mg/kg) or tacrolimus (2.4 mg/kg) alone prolonged graft survival with median survival of 14 and 13.5 days, respectively. Combined therapy of MNA (10 mg/kg) and tacrolimus 1.6 mg/kg likewise resulted in a median survival of 13.25 days and an iso-effect curve for these doses was constructed. Another iso-effect curve for median graft survival of 18-19 days, including MNA (10 mg/kg) + tacrolimus (3.2 mg/kg) in combination, MNA (30 mg/kg) alone and tacrolimus (4.8 mg/kg) alone, was constructed and both isoboles showed a straight line, demonstrating additive effects (zero interaction). In addition, histological analysis of grafts confirmed the benefit of the drug combination. No additional toxicity was noted with combined therapy. Optimal doses of MNA or tacrolimus had comparative effects on graft survival and histological changes, and a combination of the two drugs was beneficial with respect to both these parameters. The iso-effect curves verified additive effects of the drug combination.

The prevalence of gallstones in chronic liver disease is related to degree of liver dysfunction.

BACKGROUND/AIMS: Earlier studies have indicated an elevated risk for gallstone disease in patients with cirrhosis. This study aimed to evaluate the prevalence of gallstones in patients with chronic liver disease (CLD) with respect to sex, etiology, and severity of liver disease. METHODOLOGY: Four hundred and thirteen adults (176 women), mean age 51.2+/-14 years, with CLD, who had undergone liver biopsy during 1978-1993, and from whom sera were available, were investigated retrospectively. The results were compared with a population-based ultrasonography study of 556 healthy men and women, in their 40s and 60s. RESULTS: The prevalence of gallstones in patients with CLD did not differ from that in the control population. An increased frequency was observed in patients with CLD initially classified as cryptogenic, of whom the majority (60%) later were reclassified as chronic hepatitis C. The frequency of gallstones was also high in PiZ-heterozygotes for alpha1-antitrypsin deficiency (5/21, 24%) compared to non-PiZ-carriers (17/389, 4.8%), (p<0.001). In 67 patients with histologic evidence of cirrhosis, 30% (20/67) had gallstones (vs. 15% in the general population, p<0.01). The prevalence of gallstones increased significantly from Child's class A (16%) to C (56.2%). The difference was significant in males (18.2% vs. 62.5%, p=0.033), but not in females. Fifty percent of the patients with gallstones were symptomatic. CONCLUSIONS: Progressive liver dysfunction is a risk factor for gallstones particularly in males. HCV infection and PiZ carriership may further increase biliary lithogenesis.

Malononitrilamides 715 and 279 prevent accelerated cardiac allograft rejection synergistically with cyclosporin A in presensitized rats.

A77 1726 is a malononitrilarnide (MNA) and the active metabolite of leflunomide. Leflunomide has been extensively investigated and shown to be a potent immunosuppressive drug. However, the half-life of A77 1726 is about 15-18 days in humans and the leflunomide is, therefore, currently being evaluated for the treatment of autoimmune disease and not for transplantation. The search for analogues has led to the development of MNA 715 and 279, derivatives of A77 1726. Previous limited experimental experience has shown these MNAs to prevent skin allograft and xenograft rejection and graft-versus-host disease in rodents, and to reverse ongoing allograft rejection. The aim of the present study was to verify the efficacy of these MNAs in a cardiac retransplant model of sensitized rats, concerning prevention of accelerated rejection, inhibition of antibody production and interaction with cyclosporin A (CsA). Heterotopic cardiac transplantation and retransplantation in Dark Agouti (DA) to Piebald Virol Glaxo (PVG) rats was used. Subgroups of rats were given either CsA, MNA 715, MNA 279 or combined CsA/MNA for 10 days starting either day 0 or day -1, as of regrafting or no treatment. Titres of allospecific IgM and IgG were quantitated by flow cytometry. Ten days of MNA 715 or 279 from day -1 prevented accelerated rejection of the retransplant, as did CsA. Neither treatment given from day 0 prevented rejection within 24 h. However, a combination of MNA 715 or 279 and CsA from day 0 effectively prevented accelerated regraft rejection. Production of specific alloantibodies was reduced in all immunosuppressed subgroups, IgG titres at day 7 in MNA-treated subgroups being significantly lower compared with those in the CsA-treated subgroup. In conclusion, MNA 715 and 279 were shown to be potent immunosuppressants with the capacity to prevent accelerated regraft rejection in rat cardiac transplants, most efficiently in combination with CsA, and to suppress specific alloantibody production.

Feasibility and quality of biological banking of human normal and tumor tissue specimens as sources of DNA for the Malmö Diet and Cancer Study.

Human tumor and normal tissue specimens, which were collected from autopsy material 1-6 days postmortem, were compared with similar tissue specimens collected within 2 h after surgical resection and transport to the pathology department. The end point criteria used to evaluate the quality of the specimens for biological banking purposes were the extractability and yield of high molecular weight DNA and UV absorption ratios at 260:280 after collection and immediate storage of the specimens at -80 degrees C. The data demonstrated that autopsy material was a quality source of DNA, although of not such high quality as surgical biopsy specimens <2 h after resection. The advantages of using autopsy material to supplement surgical specimen collection sent to pathology, as opposed to using specimen collection at surgery wards or formalin-fixed material, as sources of DNA are: (a) large amounts of tumor and normal tissues from a variety of organ sites can be obtained without regard to the patient's health status; (b) a higher percentage of retrieval of incident cases of cancer in prospective designed trials is more likely to be achieved; and (c) the extractable DNA is of sufficiently high enough quality to permit direct analyses by molecular hybridization and sequence methodologies.

Pharmacokinetics, toxicity, side effects, receptor affinities and in vitro radiosensitizing effects of the novel metoclopramide formulations, sensamide and neu-sensamide.

Metoclopramide is a drug which has experienced worldwide use in the clinic for over 30 years as an antiemetic. Recently, it has also been shown to possess radio- and chemosensitizing properties in both animal tumour models and humans at the higher dose of 2 mg/kg. Two new metoclopramide formulations are being clinically developed and they differ mainly in whether the pH of their formulations are acidic (pH 2.5-3.5) or neutral (pH 6.5-7.0). Here we report that intramuscular administration of neutral metoclopramide is about 100% bioavailable, safer and with reduced side effects compared to acidic metoclopramide delivered by intramuscular injection to rats within the dose range of 3.5 to 14 mg/kg. The intramuscular administration of metoclopramide was also about 100% bioavailable compared to the intravenous route of administration. Furthermore, neutral metoclopramide had significantly decreased affinity for dopamine D2 receptors and increased affinity for 5-hydroxytryptamine, receptors, but the radiosensitizing potency was the same, when compared to equimolar concentrations of acidic metoclopramide. Taken together these data support the continued development of neutral metoclopramide for high dose intramuscular administration of metoclopramide for future clinical use as both an antiemetic and radiosensitizer.

Allogeneic heart transplantation activates alloreactive NK cells.

The ability of natural killer (NK) cells to recognize and reject transplants has so far been shown in hematopoietic grafts only. This study was designed to ascertain whether NK cells may also be involved in the rejection of transplanted organs. In most rat strain combinations, immunization with allogeneic cells induces a T cell response with cytotoxic T lymphocyte (CTL) activation. We have previously found one exception to this. In contrast to Wistar Furth rats (WF, RT1u), which manifest allospecific CTL activation in response to immunization with Brown Norway (BN, RT1n) cells, BN rats immunized with repeated intraperitoneal (i.p.) injections of allogeneic WF spleen cells manifest activation of alloreactive NK effector cells. The alloreactive NK cells were of the TCR-, CD3-, CD8+, and NKR-P1 intermediate phenotype and killed target cells with alloselectivity. In this study we used a heart transplantation model to study the rejection response of BN rats receiving WF grafts. NK cell infiltration was greater in WF hearts transplanted to BN recipients than in BN hearts transplanted to WF recipients. Furthermore, the extent of T cell infiltration was less in BN recipients. In WF rats transplanted with allogeneic BN hearts, CTL were activated in response to i.p. challenge with allogeneic BN cells, whereas BN rats transplanted with allogeneic WF hearts and i.p. challenged with allogeneic WF cells, manifested activation of alloreactive NK cells but no measurable activation of classic CTL. The alloreactive NK cells killed their allogeneic targets with specificity and with potency comparable to that of CTL. Furthermore, WF grafts were rejected in BN recipients as efficiently as were BN grafts in WF recipients. These results not only show cardiac allografts to be able to activate alloreactive NK cells, but also suggest that NK cells may be involved in the rejection of solid organ transplants and function as classic CTL in certain donor-recipient combinations.