P. Eschwège

Ex Vivo Fluorescence Imaging of Normal and Malignant Urothelial Cells to Enhance Early Diagnosis

Urinary cytology is a noninvasive and unconstraining technique for urothelial cancer diagnosis but lacks sensitivity for detecting low‐grade lesions. In this study, the fluorescence properties of classical Papanicolaou‐stained urothelial cytological slides from patients or from cell lines were monitored to investigate metabolic changes in normal and tumoral cells. Time‐ and spectrally‐resolved fluorescence imaging was performed at the single cell level to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by urothelial cells. The results reveal quite different fluorescence distributions between tumoral urothelial cells, characterized by a perimembrane fluorescence localization, and the normal cells which exhibit an intracellular fluorescence. This is not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H, flavoproteins or porphyrins but by various localization of the EA 50 Papanicolaou stain as revealed by both the spectral and time‐resolved parameters. The present results demonstrate that the use of single‐cell endofluorescence emission of Papanicolaou‐stained urothelial cytological slides can allow an early ex vivo diagnosis of low‐grade bladder cancers.

Surgical injuries occurring during kidney procurement performed by a renal transplantation team

ENAL surgery injuries, which sometimes occur during organ procurement, may contribute to morbidity and renal function impairment as well as increased hospital stay and cost. 1 In France, renal procurements are customarily performed by urologists directly involved in renal transplantation, who also have to deal with transplant complications. Prevention is the best way to manage renal surgery injuries, but this requires identifying the manner in which they may occur. The purpose of the present study was to clarify the causes of renal injuries during organ harvests over the past 15 years at our institution. PATIENTS AND METHODS Between 1985 and 2000 we performed 627 renal procurements (191 female and 436 male donors) including 558 from heart-beating and 69 from non– heart-beating donors. We noted the surgical injuries to the kidneys and the causes cited by physicians for not using organs. Statistical analysis was performed according to initial injury assignments based on our database. The chi-square test was used as appropriate; differences were considered statistically significantly if P .05. RESULTS A total of 1239 kidneys were harvested. Fifteen kidneys were not harvested. There were 106 kidneys not transplanted. The presence of surgical lesions was the reason for transplant refusal in four cases (1 renal decapsulation, 1 arterial injury, and 2 venous injuries, which could not be repaired properly). Twenty kidneys were refused for transplantation because they were macroscopically abnormal, namely a renal tumor or renal rupture, or because they displayed microscopic abnormalities, namely nephroangiosclerosis, glomerulosclerosis, or arteriolar injuries. Macroscopic surgical arterial injuries were observed in 2.3% (29 of 1254) of cases (26 polar artery transections and 3 hilar artery transsections). Venous lesions were observed in 0.5% (7 of 1254) of cases. Surgical injuries were more frequently observed during single-organ procurements than during multiorgan procurements: 8.2% vs 3.7%, respectively (P .05). Similarly, injuries were more frequent during non– heart-beating donor procurement: 9.3% vs 4.6% (P .01), or when the surgeon had less training (30 organ vs 30 procurements): 12% vs 3%, respectively (P .01).

Successful treatment of encrusted pyelitis in a renal transplant with local acidification and surgical ileocaliceal anastomosis.

Encrusted pyelitis was initially described in 1993. The incidence has increased with the increasing number of patients with immunodeficiency, particularly those with a renal transplant. This condition is secondary to urinary tract infection due to ureolytic microorganisms. More than 45 microorganisms have been implicated as etiological factors of encrusted cystitis and pyelitis. The treatment of encrusted cystitis or pyelitis includes specific antibiotics, urinary acidification and endoscopic excision of the calcified lesions. When treatment is not rapidly instituted, excision of the transplant is necessary. Nevertheless, in some cases the renal transplant may be surgically and medically treated. Such therapy may be beneficial to the renal transplant, which may be functional after infection. We report a case of renal transplant salvage after the development of encrusted pyelitis.

Renoprotective potency of amifostine in rat renal ischaemia–reperfusion

PURPOSE Kidneys from haemodynamically unstable donors may suffer from renal ischaemia-reperfusion (RIR) injury. RIR is associated with reactive oxygen species production that induces inflammation and activates the arachidonic acid (AA) pathway which converts AA into prostaglandin E(2). Amifostine was investigated for its renoprotective potential in RIR. MATERIALS AND METHODS The effect of amifostine (25 mg/kg  =  910 mg/m(2)) on the COX pathway, enzymatic antioxidant activity, the lipid peroxidation marker MDA, serum creatinine and apoptosis was determined in rats. Kidneys were subjected to 45 min of ischaemia and 1 or 24 h of reperfusion. Control groups (sham: coeliotomy, no ischaemia; r1: 45 min ischaemia/1 h reperfusion; r2: 45 min ischaemia/24 h reperfusion) were administered physiological saline intraperitoneally, and treated groups (E1: 45 min ischaemia/1 h reperfusion; E2: 45 min ischaemia/24 h reperfusion) received amifostine 30 min before reperfusion. RESULTS Serum creatinine increased in non-treated control rats: r1 vs sham (1.6-fold; P <0.007), r2 vs sham (2-fold; P <0.007). Amifostine decreased serum creatinine levels in treated rats: E1 vs r1 (8%; P <0.0025), E2 vs r2 (44%; P <0.0025). Amifostine reduced acute tubular necrosis (25%) 24 h after reperfusion: E1 vs r1 (P <0.004), E2 vs r2 (P <0.03) and reduced COX-2 and microsomal prostaglandin E synthase expression: E1 vs r1 (P <0.03), E2 vs r2 (P <0.02). Amifostine decreased MDA (P <0.04) and reduced caspase-3 expression but did not alter enzymatic antioxidant activity after RIR. CONCLUSIONS Amifostine decreased the degree and severity of tubular damage after reperfusion, probably by scavenging oxygen free radicals and attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.

Expression of aldehydic lipid peroxidation products in rat kidneys during warm ischemia

Abstract DURING warm ischemia (WI), liberation of metabolites such as superoxide anions and hydroxyl radicals induce severe tissue damage. 1 Stress resulting from oxygen-derived free radical is highly involved in WI damage to kidney. The main targets of these molecules are membrane lipids. 2 The two prominent lipid degradation products are malondialdehyde (MDA) and 4-hydroxynonenal (HNE). They can be detected with biochemical assays or immuno-staining using antibodies against protein-linked MDA and HNE. This article describes the study to determine qualitatively and quantitatively the liberation of MDA and HNE. These molecules measured in kidney biopsies reflect kidney damage and could be considered as significant markers especially after WI.

A fluorescence-based assay for monitoring clinical drug resistance

Background and aims Multidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor patients’ responses to chemotherapy. Methods The assay is based on the fluorescent properties of doxorubicin alone as well as in combination with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). The slide-based cell imaging technique was first optimised using a panel of breast and urothelial cancer cell lines and then extended to fine needle breast aspiration biopsy and urine cytology. Results The drug fluorescence behaviour observed on smears of clinical specimens is identical to that obtained using fixed cultured cells. The fluorescence of sensitive cells to chemotherapy is mainly localised in the nucleus, whereas resistant cells show a weak fluorescence signal localised in the cytoplasm. The difference in terms of fluorescence intensity is also highlighted through fluorescence spectra. Conclusions The results suggest that the assay provides clinically valuable information in predicting responses to doxorubicin and/or MVAC therapy. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope; as such it is technically simple, reliable and inexpensive.

Long-term results and risk factors of quadruple immunosuppression in renal transplantation

POLYCLONAL antilymphocyte globulins (ALGs) have been used in cadaver renal transplantation since 1960 for the prevention and treatment of acute allograft rejection. However, in the cyclosporine (CsA) era, their utility within multidrug immunosuppressive protocols has been debated. The rationale for biological agent induction is first to provide heavy initial immunosuppression to abrogate, delay, or reverse subsequent rejection episodes. Second, the use of ALG with delayed CsA introduction was advocated to minimize the potentially deleterious effects of CsA on cadaver transplants submitted with multiple (ie, ischemia–reperfusion) perioperative injuries. We have previously demonstrated that a quadruple association with a 14-day course of ALG, oral CsA from day 1, prednisolone, and delayed introduction of azathioprine (AZA) provided excellent results on short-term endpoints, such as 1-year graft survival, rejection incidence, and early complication incidence, without inducing overimmunosuppression complications. This study aimed to analyze long-term results (survival, complications, causes of failure, and risk factors for failure) in a cohort of 632 cadaver transplants performed at our institution from 1985 to 1997 using such protocol.