A new animal model of postsurgical bowel inflammation and fibrosis: the effect of commensal microflora.

OBJECTIVE: Ileocaecal resection (ICR) is common in Crohn's disease. Inflammation and fibrosis frequently recur at the site of anastomosis or in the small intestine (SI). No animal models of postsurgical inflammation and fibrosis exist. A model of ICR was developed in interleukin 10 (IL10) null and wild-type (WT) mice to test the hypothesis that ICR promotes postsurgical inflammation and fibrosis in the SI or anastomosis of genetically susceptible IL10 null, but not WT or germ-free (GF)-IL10 null mice. METHODS: GF-IL10 null mice were conventionalised (CONV) and 3 weeks later randomised to ICR, transection (T) or no treatment (NoTx). Age-matched conventionally raised (CONV) WT and GF-IL10 null mice received ICR, T or NoTx. Animals were killed 28 days later. Histological scoring, real-time PCR for tumour necrosis factor alpha and collagen, and immunostaining for CD3(+) T cells assessed inflammation and fibrosis. RESULTS: After ICR, CONV-IL10 null, but not CONV-WT mice, developed significant inflammation and fibrosis in the SI and inflammation in anastomosis compared with NoTx or T controls. Fibrosis occurred in the anastomosis of both CONV-IL10 null and CONV-WT mice following ICR. GF-IL10 null mice developed little or no inflammation or fibrosis in the SI or anastomosis after ICR. CONCLUSIONS: ICR in CONV-IL10 null mice provides a new animal model of postsurgical inflammation and fibrosis in the SI and anastomosis. Absence of inflammation and fibrosis in the SI of CONV-WT and GF-IL10 null mice following ICR indicates that postsurgical small bowel disease occurs only in genetically susceptible IL10 null mice and is bacteria dependent.

Hypothesis: Bacterial induced inflammation disrupts the orderly progression of the stem cell hierarchy and has a role in the pathogenesis of breast cancer.

BACKGROUND: The hierarchical model of stem cell genesis is based on the idea that the number of cell divisions between the zygote and fully differentiated epithelial cells is kept close to the minimum, which is log to the base 2 of the total number of cells produced in a human lifetime. The model assumes the orderly progression of stem cell divisions requires precise control at every stage in development. If the orderly progression is maintained then cancer will be rare. A prediction of the model is that if the orderly progression of the stem cell hierarchy is disturbed by trauma, ulceration or inflammation then cancer will occur. HYPOTHESIS: Bacterial induced inflammation in breast ducts disturbs the stem cell hierarchy and is a cause of breast cancer. EVIDENCE: Mammalian milk is not sterile. It contains a range of bacteria, derived endogenously by the entero-mammary circulation. The dominant flora consists of lactose fermenting bacteria. Pregnancy and breast feeding reduce the risk of subsequent breast cancer. The implication is that a lactose fermenting bacterial flora in breast ducts is protective. Malignant and benign breast tissue contains bacteria derived endogenously, but studies so far have not revealed a specific flora associated with malignancy. Periodontitis is associated with oral, oesophageal, colonic, pancreatic, prostatic and breast cancer. The pathogenic bacteria which cause periodontitis spread endogenously to cause inflammation at other epithelial sites. Meta-analysis of epidemiological studies shows that the consumption of yoghurt is associated with a reduction in the risk of breast cancer. CONCLUSION: The hypothesis, although not proven, is supported by the available evidence. Lactose fermenting bacteria protect but pathogenic bacteria which induce inflammation raise the risk of breast cancer. The consumption of yoghurt also appears to be protective.

Suppressor of cytokine signaling 3 (SOCS3) limits damage-induced crypt hyper-proliferation and inflammation-associated tumorigenesis in the colon.

Intestinal injury or chronic inflammation induce cytokines that promote crypt regeneration and mucosal repair. If excessive or prolonged, such mechanisms may increase colon cancer risk. Factors that terminate or limit cytokine action in intestinal epithelial cells (IEC) may protect against crypt hyperplasia and neoplasia. We hypothesized that suppressor of cytokine signaling-3 (SOCS3) is such a factor. Mice with Vilin-promoter/Cre-recombinase (VC)-mediated IEC-specific SOCS3 gene disruption (VC/HO), WT/HO littermates with floxed but intact SOCS3 genes and VC/WT mice were studied. Colon was examined after acute dextran sodium sulfate (DSS)-induced mucosal injury or after azoxymethane (AOM) and chronic DSS. Signaling pathways were examined in colon, cultured IEC or colon cancer cell lines. VC/HO mice showed no basal phenotype. After acute DSS, VC/HO exhibited enhanced crypt proliferation and crypt hyperplasia and reduced transforming growth factor (TGF) beta expression in colon. Inflammation and mucosal damage were similar across genotypes. Following AOM/DSS, VC/HO mice had increased size, number and load of colonic tumors and increased STAT3 and nuclear factor-kappa B (NF-kappaB) activation in colon. In vitro, SOCS3 overexpression reduced proliferation, IL-6-mediated STAT3 activation and tumor necrosis factor (TNF) alpha-mediated NF-kappaB activation. We conclude that cytokine induction of SOCS3 normally provides an intrinsic mechanism to limit injury-induced crypt hyperproliferation and inflammation-associated colon cancer by regulating both STAT3 and NF-kappaB pathways.

The kinetics of mycophenolic acid and its glucuronide metabolite in adult kidney transplant recipients.

BACKGROUND: Mycophenolic acid kinetics have been reported to vary after renal transplantation, and mycophenolic acid area under the concentration-time curve (AUC) is the best predictor of suppression of graft rejection. METHODS: To determine whether mycophenolic acid kinetics vary after renal transplantation and to examine the potential role of enterohepatic recirculation, we investigated the kinetics of mycophenolic acid and mycophenolic acid glucuronide on days 2, 5, and 28 after transplantation in 10 kidney transplant recipients (male/female ratio, 1.5; mean age, 41.7 +/- 5.0 years) given 1 g mycophenolate mofetil twice a day. To facilitate therapeutic drug monitoring, we examined a limited sampling strategy for estimating 12-hour mycophenolic acid [AUC(0-12)]. RESULTS: The mean +/- SE AUC(0-12) for mycophenolic acid on day 28 was 38.5 +/- 1.6 mg x h/L, with a secondary peak 4 to 8 hours after dosing that was attributable to enterohepatic recirculation. Marked variability was shown in the kinetic profile of mycophenolic acid among patients across the three sampling days. Mycophenolic acid AUC(0-12) was positively predicted by both serum creatinine (P = .01) and serum albumin (P = .03) but not by time after transplantation, body weight, or trough concentration. Limited sampling (at 0, 1, 3, and 6 hours) accounted for 84.1% of the variability in the mycophenolic acid AUC(0-12) data and predicted the AUC(0-12) closely (r2 = 0.954) when evaluated in 10 different kidney transplant recipients. CONCLUSIONS: Mycophenolic acid AUC(0-12) is predicted by serum albumin and creatinine after kidney transplantation, and the AUC(0-12) may be determined during the early posttransplant period while the patient remains hospitalized with use of a limited sampling strategy to facilitate therapeutic drug monitoring.

Effect of OKT3 in steroid-resistant renal transplant rejection.

Between January 1, 1982, and November 1, 1986, 169 cadaver renal graft transplantations were performed at this hospital with CsA as induction therapy. OKT3 was not available in this period. Of these grafts, 15.9% were lost within 6 months, 10.7% from acute rejection (AR). Between November 1, 1986, and October 1, 1992, 483 cadaver renal graft transplantation were performed. Induction therapy included CsA and OKT3 was available. Of these grafts, 8.7% were lost inside 6 months, 3.1% from AR. Of these last 483 grafts, 113 received 125 courses of OKT3. Ten courses were prophylactic, and 115 courses in 103 patients were for rejection resistant to steroid therapy (biopsy proven in all but 2 cases. Ninety-three percent of rejection episodes treated with OKT3 responded, at least initially. Graft survival in OKT3-treated patients was 81%, 77%, and 76% at 6 months, 1 year, and 2 years, respectively. In contrast, graft survival in steroid-resistant rejection during the first period (without OKT3) was 59%, 57%, and 57% at these intervals. There were 8 infective deaths within 6 months in the 113 OKT3-treated patients, compared with 2 in the 343 who did not receive OKT3 (P < 0.001). There were 7 viral deaths in the OKT3 group compared with none in those not receiving OKT3 (P < 0.001). Prophylaxis with oral acyclovir and cotrimoxazole was instituted in October 1990 in OKT3-treated patients and ganciclovir use was increased. Since this change, no further viral deaths have occurred. OKT3 is a very effective antirejection agent, but its use is associated with an increased mortality from viral infections. With appropriate prophylaxis and treatment, however, this mortality can be reduced.

A comparison of the effects of dialysis and renal transplantation on the survival of older uremic patients.

BACKGROUND: Patients over age 60 constitute half of all new patients accepted into the renal replacement therapy programs in Australia. However, the optimal treatment of their end-stage renal disease remains controversial. The aim of the present study was to compare survival for dialysis and renal transplantation in older patients who were rigorously screened and considered eligible for transplantation. METHODS: The study cohort consisted of 174 consecutive patients over 60 who were accepted on to the Queensland cadaveric renal transplant waiting list between January 1, 1993 and December 31, 1997. Follow-up was terminated on October 1, 1998. Data were analyzed on an intention-to-transplant basis using a Cox regression model with time-varying explanatory variables. An alternative survival analysis was also performed, in which patients no longer considered suitable for transplantation were censored at the time of their removal from the waiting list. RESULTS: There were 67 patients receiving a renal transplant, whereas the other 107 continued to undergo dialysis. These two groups were well matched at baseline with respect to age, gender, body mass index, renal disease etiology, comorbid illnesses, and dialysis duration and modality. The overall mortality rate was 0.096 per patient-year (0.131 for dialysis and 0.029 for transplant, P<0.001). Respective 1-, 3- and 5-year survivals were 92%, 62%, and 27% for the dialysis group and 98%, 95%, and 90% (P<0.01) for the transplant group. Patients in the transplant group had an adjusted hazard ratio 0.16 times that of the dialysis group (95% confidence interval 0.06-0.42). If patients were censored at the time of their withdrawal from the transplant waiting list, the adjusted hazard ratio was 0.24 (95% confidence interval 0.09-0.69). CONCLUSIONS: Renal transplantation seems to confer a substantial survival advantage over dialysis in patients with end-stage renal failure who are rigorously screened and considered suitable for renal transplantation.

A prospective randomized trial of low-dose versus high-dose steroids in cadaveric renal transplantation.

Low-dose steroid regimens, in combination with azathioprine, have become increasingly common for immunosuppression of renal transplant recipients. The change from conventional high-dose steroid regimens was prompted by the results of several prospective trials that showed similar graft survivals with high-dose and low-dose steroids, but a lower incidence of steroid-induced complications in low-dose-steroid--treated patients. However, the number of patients entered into the trials was small, and consequently there remained a possibility that a clinically relevant difference in graft survival could have remained undetected. A multi-center prospective trial was performed to compare graft survival with high-dose (91 patients) and low-dose (98 patients) oral steroids in combination with azathioprine. There was significantly worse graft survival in the low-dose group. The difference was largely due to a poor graft survival in patients receiving low-dose steroids and azathioprine less than 1.75 mg/kg/day. Graft survivals were similar in the high-dose and low-dose steroid patients who received azathioprine doses of greater than 1.75 mg/kg/day. The results indicate that the combination of low doses of both steroids and azathioprine provides inadequate immunosuppression in renal transplantation, although higher doses of azathioprine allow the use of low-dose steroids without significantly more graft losses than with high-dose steroids.

Novel surveillance and cure of a donor-transmitted lymphoma in a renal allograft recipient.

BACKGROUND: In this report we describe a malignant lymphoma of donor origin inadvertently transplanted into two renal allograft recipients, despite standard comprehensive donor screening. The successful clearance of the tumor from both patients and a novel method of surveillance are detailed. METHODS: Initial management consisted of withdrawal of immunosuppression to promote rejection of the allograft and the transplanted tumor in both patients, followed by graft removal. Peripheral blood microchimerism was assessed in both recipients using nested polymerase chain reaction to detect the DYZ3 gene on the Y chromosome (donor male, recipients female). RESULTS: Although microchimerism was detected on day 6 after transplantation and day 1 after explantation, repeat peripheral blood examination at 1, 3, and 6 months after explantation demonstrated no microchimerism. Both patients remain well at 12 months and have been relisted for transplantation. CONCLUSION: Despite inadvertent transplantation of a previously undiagnosed malignancy of donor origin, the recipients' immune response was able to eliminate donor tumor cells after the withdrawal of immunosuppression. Repeated surveillance of peripheral blood from both recipients, using a novel application of the technique of nested polymerase chain reaction to amplify donor DNA, demonstrated no persistence of donor cells, supporting effective eradication of the donor malignancy.

A retrospective analysis of mycophenolic acid and cyclosporin concentrations with acute rejection in renal transplant recipients.

OBJECTIVES: Although monitoring of cyclosporin (CsA) is standard clinical practice postrenal transplantation, mycophenolic acid (MPA) concentrations are not routinely measured. There is evidence that a relationship exists between MPA area under the concentration-time curve (AUC) and rejection. In this study, a retrospective analysis was undertaken of 27 adult renal transplant recipients. METHODS: Patients received CsA and MPA therapy and had a four-point MPA AUC investigation. The relationship between MPA AUC performed in the first week after transplantation, as well as median trough cyclosporin concentrations, and clinical outcomes in the first month posttransplant were evaluated. RESULTS: A total of 12 patients experienced biopsy proven rejection (44.4%) and 4 patients had gastrointestinal adverse events (14.8%). A statistically significant relationship was observed between the incidence of biopsy proven rejection and both MPA AUC (p = 0.02) and median trough CsA concentration (p = 0.008). No relationship between trough MPA concentration and rejection was observed (p = 0.21). Only 3 of 11 (27%) patients with an MPA AUC > 30 mg x h/L and a median trough CsA > 175 microg/L experienced acute rejection, compared with a 56% incidence of rejection for the remaining 16 patients. Patients who experienced adverse gastrointestinal events had significantly lower MPA AUC (p = 0.04), but median trough CsA concentrations were not significantly different (p = 0.24). Further, 3 of these 4 patients had rejection episodes. CONCLUSIONS: In addition to standard CsA monitoring, we propose further investigation of the use of a 4-point sampling strategy to predict MPA AUC in the first week posttransplant, which may facilitate optimization of mycophenolate mofetil dose at a time when patients are most vulnerable to acute rejection.

Severe tubulo-interstitial disease in a renal allograft due to cytomegalovirus infection.

A 34 year old female developed impaired function of her renal allograft 21 months post-transplant. This was associated with lethargy, pyrexia, tenosynovitis, pancytopenia and a colonic ulcer. Severe tubulo-interstitial changes with intranuclear inclusion bodies and intracytoplasmic herpes type viral particles were seen on renal biopsy. There was no evidence of rejection. Cytomegalovirus was cultured from the urine and there was a rise in CMV antibody titer. These findings suggested the renal impairment was due to a direct cytopathic effect of the CMV. Despite treatment with transfer factor and adenine arabinoside, there was progressive loss of graft function.

The role of DEXA bone densitometry in evaluating renal osteodystrophy in continuous ambulatory peritoneal dialysis patients.

OBJECTIVE: The aims of this study were to assess the clinical utility of total and regional bone densitometry in a large continuous ambulatory peritoneal dialysis (CAPD) population and to determine the clinical, biochemical, and radiographic variables that best identified osteopenic CAPD patients. DESIGN AND PATIENTS: A cross-sectional study was performed on 45 CAPD patients (19 males, 26 females), comprising the total CAPD population at the Princess Alexandra Hospital. MAIN OUTCOME MEASURES: Total body (TB), anteroposterior lumbar spine (APL), femoral neck (FN), Ward's triangle (WT), and skull bone mineral densities (BMDs) were measured using dual-energy x-ray absorptiometry (DEXA) and then correlated with clinical, biochemical, and radiographic indices of uremic osteodystrophy. RESULTS: BMDs were not significantly different from age- and sex-matched reference population data. Considerable regional variation of BMD Z scores were noted between FN (-0.11 +/- 0.23), WT (-0.11 +/- 0.22), and APL (1.22 +/- 0.04) (p = 0.003). APLZ scores were significantly reduced in patients with a previous history of fracture (-1.36 +/- 1.07 vs 0.89 +/- 0.31), bone pain (-0.72 +/- 1.08 vs 1.01 +/- 0.31), or steroid treatment (-0.62 +/- 0.39 vs 1.16 +/- 0.35). Increased BMD Z scores for APL (1.82 +/- 0.57 vs 0.38 +/- 0.29, p < 0.05), FN (0.32 +/- 0.36 vs -0.38 +/- 0.29, p = 0.014), and WT (0.45 +/- 0.38 vs -0.45 +/- 0.26, p < 0.05) were found in patients with radiographic hyperparathyroid bone disease. Both APL BMD Z scores and skull BMDs were weakly correlated with PTH (r = -0.33, p < 0.05 and r = -0.33, p < 0.05, respectively) and with CAPD duration (r = 0.30, p < 0.05 and r = -0.30, p < 0.05). Generally, however, total body and regional BMDs were poorly related to age, renal disease type, dialysis duration, renal failure duration, serum aluminum, calcium, phosphate, alkaline phosphatase, osteocalcin, and parathyroid hormone. CONCLUSIONS: We conclude that the prevalence of osteopenia is not increased in CAPD patients. Clinical and biochemical parameters do not reliably predict BMD measurements, but prior steroids and bone symptoms are major risk factors for important bone loss. Although DEXA can reliably detect osteopenia in different skeletal regions, its usefulness in detecting osteodystrophy is limited by the confounding effects of superimposed hyperparathyroid osteosclerosis, which increases BMD.

Serum leptin correlates with fat mass but not dietary energy intake in continuous ambulatory peritoneal dialysis patients.

OBJECTIVES: In view of previous studies demonstrating hyperleptinemia in uremic and hemodialysis patients, the aims of the present study were to determine whether serum leptin levels are elevated in peritoneal dialysis (PD) patients, to establish whether leptin is significantly removed by PD, and to elucidate the relationship of plasma leptin to body composition, dietary intake, nutritional indices, and dialysis adequacy. DESIGN: Cross-sectional analysis of PD patients and matched healthy controls. SETTING: Tertiary-care institutional dialysis center. PARTICIPANTS: The study included 49 PD patients [35 women and 14 men; median age 63 years, interquartile range (IQR) 49.5-68.5 yr; body mass index (BMI) 25.5 +/- 0.8] and 27 controls (11 men and 16 women; median age 42 years, IQR 34.8-51; BMI 27.2 +/- 0.9). For evaluation of leptin clearance, 8 patients receiving nocturnal intermittent PD were also evaluated. MAIN OUTCOME MEASURES: The primary outcome measure was plasma leptin concentration. Dialysate leptin concentration was also measured in 7 patients. RESULTS: Serum leptin levels were significantly higher (p < 0.01) in patients (males: median 11 ng/mL, IQR 9-19 ng/mL; females: 53 ng/mL, 19.5-128 ng/mL) compared with controls (males: 5.5 ng/mL, 4-9.5 ng/mL; females: 12 ng/mL, 9.8-17.3 ng/mL). Leptin levels in both groups correlated positively with BMI (r = 0.64 and 0.60, respectively; p < 0.0001) and with percentage body fat determined by dual-energy x-ray absorptiometry (r = 0.86 and 0.82, respectively; p < 0.01). Dialysis patients exhibited a greater increase in serum leptin for any given increase in BMI. No significant correlation was observed between leptin concentration and residual renal function, dialysis adequacy (Kt/V), dietary protein or caloric intake, or serum levels of albumin, prealbumin, C-reactive protein, glucose, and insulin-like growth factor-I. Although leptin was detectable in peritoneal dialysate after a 6-hour dwell (median 4.2 ng/mL, IQR 1.1-8.5 ng/mL, n = 8), serum leptin levels were not appreciably lowered following intermittent PD via an automated cycler (63.9 +/- 19.3 ng/mL vs 57.6 +/- 20.5 ng/mL, p = NS, n = 8). CONCLUSIONS: Serum leptin levels are elevated in PD patients and are not appreciably cleared by PD. Although hyperleptinemia correlates poorly with dialysis adequacy and protein intake, a strong and significant relationship was maintained between serum leptin and fat mass. Serum leptin could therefore serve as a useful clinical marker of body fat content in PD patients.

Is obesity a favorable prognostic factor in peritoneal dialysis patients?

OBJECTIVE: To determine the influence of an elevated body mass index (BMI) on cardiovascular outcomes and survival in peritoneal dialysis (PD) patients. DESIGN: Prospective, observational study of a prevalent PD cohort at a single center. SETTING: Tertiary care institutional dialysis center. PATIENTS: The study included all patients with a BMI of at least 20 who had been receiving PD for at least 1 month as of 31 January 1996 (n = 43). Patients were classified as overweight [BMI > 27.5; mean +/- standard error of mean (SEM): 32.1 +/- 1.1; n = 14] or normal weight (BMI 20-27.5; mean +/- SEM: 23.8 +/- 0.4; n = 29). OUTCOME MEASURES: Patient survival and adverse cardiovascular events (myocardial infarction, congestive cardiac failure, cerebrovascular accident, and symptomatic peripheral vascular disease) were recorded over a 3-year period. RESULTS: At baseline, no significant differences were seen between the groups in clinical, biochemical, nutritional, or echocardiographic parameters, except for a lower dietary protein intake (0.97 +/- 0.10 g/kg/day vs 1.44 +/- 0.10 g/kg/day, p = 0.004) and a higher proportion of well-nourished patients by subjective global assessment (100% vs 72%, p < 0.05) in the overweight group. After 3 years of follow-up, 29% of overweight patients and 69% of normal-weight patients had died (p < 0.05). Using a Cox proportional hazards model, a BMI greater than 27.5 was shown to be an independent positive predictor of patient survival, with an adjusted hazard ratio (HR) of 0.09 [95% confidence interval (CI): 0.01-0.85; p < 0.05]. However, being overweight did not significantly influence myocardial infarction-free survival (adjusted HR: 0.33; 95% CI: 0.07-1.48; p = 0.15) or combined adverse cardiovascular event-free survival (adjusted HR: 0.67; 95% CI: 0.23-1.93; p = 0.46). CONCLUSIONS: Obesity conferred a significant survival advantage in our PD population. Obese patients should therefore not be discouraged from receiving PD purely on the basis of BMI. Moreover, maintaining a higher-than-average BMI to preserve "nutritional reserve" may help to reduce the mortality and morbidity rates associated with PD.