Pulsed-light inactivation of pathogenic and spoilage bacteria on cheese surface.

Cheese products are susceptible to postprocessing cross-contamination by bacterial surface contamination during slicing, handling, or packaging, which can lead to food safety issues and significant losses due to spoilage. This study examined the effectiveness of pulsed-light (PL) treatment on the inactivation of the spoilage microorganism Pseudomonas fluorescens, the nonenterohemorrhagic Escherichia coli ATCC 25922 (nonpathogenic surrogate of Escherichia coli O157:H7), and Listeria innocua (nonpathogenic surrogate of Listeria monocytogenes) on cheese surface. The effects of inoculum level and cheese surface topography and the presence of clear polyethylene packaging were evaluated in a full factorial experimental design. The challenge microorganisms were grown to early stationary phase and subsequently diluted to reach initial inoculum levels of either 5 or 7 log cfu/slice. White Cheddar and process cheeses were cut into 2.5×5 cm slices, which were spot-inoculated with 100 µL of bacterial suspension. Inoculated cheese samples were exposed to PL doses of 1.02 to 12.29 J/cm(2). Recovered survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. The PL treatments were performed in triplicate and data were analyzed using a general linear model. Listeria innocua was the least sensitive to PL treatment, with a maximum inactivation level of 3.37±0.2 log, followed by P. fluorescens, with a maximum inactivation of 3.74±0.8 log. Escherichia coli was the most sensitive to PL, with a maximum reduction of 5.41±0.1 log. All PL inactivation curves were nonlinear, and inactivation reached a plateau after 3 pulses (3.07 J/cm(2)). The PL treatments through UV-transparent packaging and without packaging consistently resulted in similar inactivation levels. This study demonstrates that PL has strong potential for decontamination of the cheese surface.

Analysis of patients treated with living pig tissue for evidence of infection by porcine endogenous retroviruses.

The use of pigs as a source of cells and organs for transplantation has the potential to reduce the current chronic shortage of organs for the treatment of many end-stage diseases. The risk of transmission of infectious agents across the species barrier (zoonoses) has to be assessed. Many such agents can be eliminated from the pig herd. However, porcine endogenous retroviruses, which are carried within the pig genome, are not easily eliminated. They can infect primary and immortalized human cells in vitro, but to date no evidence for in vivo infection has been found in retrospective studies of humans exposed to viable porcine cells. Small-scale clinical trials using porcine cells for the treatment of Parkinson's and Huntington's disease are currently in progress. The prospective monitoring of these patients in conjunction with further research into the biology of this virus will help address safety issues.

Pharmacokinetics and tolerability of 40-0-[2-hydroxyethyl]rapamycin in de novo liver transplant recipients.

BACKGROUND: 40-0-[2-Hydroxyethyl]rapamycin (RAD), a novel macrolide with potent immunosuppressive and antiproliferative activities, prevents rejection in animal allotransplantation models. This phase I trial assessed the effects of bile diversion, administration route, and time after transplant on RAD pharmacokinetics after single-dose administration in de novo liver allograft recipients. The influence of RAD on cyclosporine (CsA) pharmacokinetics and the safety of RAD were also evaluated. METHODS: Twenty-six de novo liver allograft recipients were assigned to one of four treatment groups based on the presence or absence of a T tube, administration route (nasogastric or nasoduodenal), and timing of RAD administration. Patients received a single 7.5-mg RAD dose on one to three occasions in addition to CsA (Neoral) and corticosteroids. Steady-state cyclosporine profiles with and without RAD coadministration were evaluated. Results. Recipients with bile diversion demonstrated lower peak concentration (Cmax) than those without, but overall drug exposure (AUC) was not altered. Cmax and AUC were not influenced by administration route. A trend towards higher Cmax on postoperative day 3 than on postoperative day 1 was noted, although AUC was not altered. Single-dose RAD coadministration did not affect steady-state CsA pharmacokinetics. RAD was well tolerated and caused few drug-related adverse effects. RAD administration did not increase infection rates or produce clinically significant changes in laboratory parameters. Conclusions. In de novo liver transplant recipients, the overall extent of RAD absorption was not influenced by bile diversion, administration route, or time of administration. CsA pharmacokinetics were not affected by single-dose RAD coadministration. RAD capsules administered in single doses of 7.5 mg were well tolerated and safe.

NOF-11: a one-year pediatric randomized double-blind comparison of neoral versus sandimmune in orthotopic liver transplantation.

BACKGROUND: Although cyclosporine (CsA) has been a mainstay in liver transplantation immunosuppression the original formulation [Sandimmune (SIM)] has variable absorption, particularly in children. Neoral is a new formulation of CsA that may have improved biovailability that would be advantageous in children. This study was undertaken to assess the pharmacokinetics (PK) and effects on outcome of Neoral versus Sandimmune (SIM) in primary pediatric liver transplant recipients. METHODS: Thirty-two patients were randomized to receive Neoral (17 patients) or SIM (15 patients) in the early posttransplant period (days 1-7) in a double-blind fashion. Intravenous CsA was instituted immediately posttransplant followed by Neoral or SIM as soon as the patient was tolerating oral fluids (days 1-7). PK were compared after the first dose (1-7 days), 3 weeks, and 6 and 12 months posttransplant. In addition, side effects, effect of age and food on absorption, and rejection episodes were assessed by intent to treat analysis. Notable characteristics of this study include the use of a central laboratory for all sample analyses and the assessment of renal function using radioisotopic evaluation of glomerular filtration rates. RESULTS: At baseline the two groups were comparable. Neoral resulted in higher peak levels of CsA and total drug exposure with comparable time to peak drug levels at days 1-7 and week 3. This trend was maintained at 6 and 12 months. Time on i.v. CsA was reduced in the Neoral group (8.4 vs. 11.1 days) and the weight adjusted daily dose of SIM required to achieve target trough levels was about 2-fold more than Neoral from day 22 onward. In addition, biopsy proven and treated and steroid-resistant rejection episodes were fewer in the Neoral group (6 vs. 12; P=0.01 and 1 vs. 8: P=0.004, respectively). Side effects were comparable in both treatment groups. CONCLUSIONS: Neoral was well tolerated and had greater biovailability than SIM without any increase in the incidence of side effects. In addition fewer episodes of rejection were observed with Neoral versus SIM. We conclude that Neoral is the CsA formulation of choice for use in pediatric liver transplant recipients.

Entry-into-human study with the novel immunosuppressant SDZ RAD in stable renal transplant recipients.

AIMS: To evaluate the tolerability of single oral SDZ RAD doses in stable renal transplant recipients and the pharmacokinetics of ascending SDZ RAD doses when coadministered with steady-state cyclosporin A microemulsion (Neoral). METHODS: This randomized, double-blind, placebo-controlled, sequential study involved 54 patients in six treatment groups; a different SDZ RAD dose (0.25, 0. 75, 2.5, 7.5, 15, 25 mg) was assessed in each group. Patients received a single oral dose of SDZ RAD (n=6) or placebo (n=3) with their usual Neoral dose. SDZ RAD and cyclosporin A pharmacokinetic parameters were determined. RESULTS: All SDZ RAD doses were well tolerated, with no discontinuations due to adverse events, serious adverse events, or deaths. Similar proportions of patients receiving SDZ RAD and placebo had at least one adverse event (44% and 50%, respectively). Mean changes in laboratory variables (baseline to endpoint) showed no clinically meaningful differences between SDZ RAD and placebo groups. SDZ RAD was absorbed rapidly and showed dose-proportional pharmacokinetics (dose: 2.5-25 mg), based on systemic exposure. Multiple postabsorptive phases in the pharmacokinetic profile indicate tissue distribution. The elimination half-life ranged from 24 to 35 h across the five highest dose groups. Pharmacokinetics were similar in men and women. Co-administration of escalating single oral SDZ RAD doses did not affect steady-state cyclosporin A pharmacokinetics. CONCLUSIONS: SDZ RAD was well tolerated; safety profiles of SDZ RAD and placebo were similar. SDZ RAD pharmacokinetics were dose-proportional across the range 2.5-25 mg in conjunction with cyclosporin A-based therapy, according to systemic exposure. Cyclosporin A pharmacokinetics were not affected by coadministration of single oral doses of 0.25-25 mg SDZ RAD.

Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group.

Pig organs may offer a solution to the shortage of human donor organs for transplantation, but concerns remain about possible cross-species transmission of porcine endogenous retrovirus (PERV). Samples were collected from 160 patients who had been treated with various living pig tissues up to 12 years earlier. Reverse transcription-polymerase chain reaction (RT-PCR) and protein immunoblot analyses were performed on serum from all 160 patients. No viremia was detected in any patient. Peripheral blood mononuclear cells from 159 of the patients were analyzed by PCR using PERV-specific primers. No PERV infection was detected in any of the patients from whom sufficient DNA was extracted to allow complete PCR analysis (97 percent of the patients). Persistent microchimerism (presence of donor cells in the recipient) was observed in 23 patients for up to 8.5 years.

The kidney in children with tyrosinemia: sonographic, CT and biochemical findings.

BACKGROUND: Tyrosinemia relates to a deficiency of fumarylacetoacetate hydrolase and presents early in life with central nervous system and liver abnormalities. Renal function is often impaired. Little is known about the architecture and function of the kidneys. OBJECTIVE: Imaging changes on US and CT are compared to the function of the kidneys in children with tyrosinemia, and followed after liver transplantation. MATERIALS AND METHODS: Renal sonography, CT and renal function tests in 32 children were reviewed. Renal length, volume, echogenicity and nephrocalcinosis were evaluated. Renal function was assessed by glomerular filtration rate, and the presence of aminoaciduria, acidosis and calciuria. Seventeen children had open renal biopsy during time of liver transplantation. Histology was reviewed. Statistical analyses relating renal structure to function were performed, and repeated after transplantation. RESULTS: The kidneys were enlarged (47 %), hyperechogenic (47 %) and showed nephrocalcinosis (16 %). There was delayed excretion of contrast medium at CT in 64 %. Aminoaciduria was present in 82 % of children, hypercalciuria in 67 %, tubular acidosis in 59 %, and low GFR in 48 %. Delayed excretion of contrast was associated with low GFR (P < 0.05). Renal biopsies showed dilated tubules (81 %), interstitial fibrosis (56 %), glomerulosclerosis (56 %) and tubular atrophy (56 %). During a mean observation period of 3 years following liver transplantation, GFR improved in 50 %, tubular acidosis in 50 % and hypercalciuria in 70 %. No change was noted in renal size or sonographic architecture. CONCLUSION: Renal architecture and function are abnormal in the majority of children with tyrosinemia. Liver transplantation improves renal function in about 50 % of patients, but abnormal renal size and architecture persist.

Bile duct cells: a novel in vitro model for the study of lipid metabolism and bile acid production.

Immortalized bile duct cells (BDC), derived from transgenic mice harboring the SV40 thermosensitive immortalizing mutant gene ts458, were utilized to investigate the role of the biliary epithelium in lipid and sterol metabolism. This cell model closely resembles the in vivo situation because it expresses the specific phenotypic marker cytokeratin 19 (CK-19), exhibits the formation of bile duct-like structures, and displays well-formed microvilli projected from the apical side to central lumen. The BDC were found to incorporate [14C]oleic acid (in nmol/mg protein) into triglycerides (121 +/- 6), phospholipids (PL; 59 +/- 3), and cholesteryl ester (16 +/- 1). The medium lipid content represented 5.90 +/- 0.16% (P < 0. 005) of the total intracellular production, indicating a limited lipid export capacity. Analysis of PL composition demonstrated the synthesis of all classes of polar lipids, with phosphatidylcholine and phosphatidylethanolamine accounting for 60 +/- 1 and 24 +/- 1%, respectively, of the total. Differences in PL distribution were apparent between cells and media. Substantial cholesterol synthesis was observed in BDC, as determined by the incorporation of [14C]acetate suggesting the presence of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. With the use of [14C]acetate and [14C]cholesterol as precursors, both tauro- and glycoconjugates of bile acids were synthesized, indicating the presence of cholesterol 7alpha- and 26R-hydroxylases, the key enzymes involved in bile acid formation. The transport of bile acids was not limited, as shown by their marked accumulation in the medium (>6-fold of cell content). HMG-CoA reductase (53.0 +/- 6.7), cholesterol 7alpha-hydroxylase (15. 5 +/- 0.5), and acyl-CoA:cholesterol acyltransferase (ACAT; 201.7 +/- 10.2) activities (in pmol. min-1. mg protein-1) were present in the microsomal fractions. Our data show that biliary epithelial cells actively synthesize lipids and may directly contribute bile acids to the biliary fluid in vivo. This BDC line thus represents an efficient experimental tool to evaluate biliary epithelium sterol metabolism and to study biliary physiology.

Randomized comparison of ganciclovir plus intravenous immune globulin (IVIG) with IVIG alone for prevention of primary cytomegalovirus disease in children receiving liver transplants.

A randomized placebo-controlled trial was conducted to determine the benefit of ganciclovir (5 mg/[kg x d]) for 30 days in addition to intravenous immune globulin (IVIG) for 16 weeks for prevention of primary cytomegalovirus (CMV) disease in children receiving liver transplants. Patients were monitored for 6 months after transplantation. The two groups of patients (recipients of 29 ganciclovir plus IVIG and 27 recipients of IVIG alone) were similar in terms of age, sex, and underlying disease. The incidence of CMV disease among the ganciclovir plus IVIG recipients and the IVIG alone recipients was 17% and 26%, respectively, and the time to disease in these recipients was 46 days and 32 days, respectively. There was no difference between groups in terms of survival; episodes of rejection, bacteremia, or fungemia; use of immunosuppressive agents; and incidence of leukopenia or thrombocytopenia. These results suggest that a 4-week course of ganciclovir with IVIG is not more effective than IVIG alone for prevention of primary CMV disease. Since short-term prophylaxis with ganciclovir may delay the onset of CMV disease, further studies with a longer course of ganciclovir prophylaxis are warranted.

Developmental profile of mitochondrial glycine N-acyltransferase in human liver.

OBJECTIVE: To study the developmental profile of glycine N-acyltransferase (GAT) in the livers of children of various ages and to compare the total and specific GAT activity with that of the adult control subjects. METHODS: We measured the specific and the total mitochondrial activity of GAT in liver samples taken from 13 children 4 hours to 11 years of age. The samples were compared with those of control adults aged 24 to 40 years. Samples, either from liver-transplant donors or from autopsy, from those who died of a disorder not related to the liver, were obtained between 6 and 36 hours after death. RESULTS: At 4 hours after birth, very low specific activity and the total liver mitochondrial activity were observed (0.19 mumol/min per milligram protein and 210 mumol/min), with a steady increase up to age 7 months (2.51 mumol/min per milligram protein and 812 mumol/min). The mean specific and total GAT activity in children (n = 5) aged 18 months to 11 years was 6.38 +/- 0.13 and 1389 +/- 43 and in control adults aged 24 to 40 years (n = 3) was 6.5 +/- 0.3 and 1461 +/- 71 mumol/min per milligram protein and mumol/min, respectively. These specific and total GAT activity values from children aged 18 months to 11 years were not statistically significant (by analysis of variance and Mann-Whitney test) in comparison with the corresponding activity values from the adult control subjects. CONCLUSIONS: Our results indicate that up to age 7 months, children have only 5% to 40% of liver GAT-specific activity, whereas the peak activity is achieved at 18 months and remains constant until age 40 years. The delayed development of GAT in children may thus compromise the detoxification of various drugs and xenobiotics.

Ursodeoxycholic acid improves the hepatic metabolism of essential fatty acids and retinol in children with cystic fibrosis.

OBJECTIVE: Several clinical trials of ursodeoxycholic acid (UDCA) have shown improvement of liver-function test results in cystic fibrosis (CF) with liver disease; however, there is no evidence that the long-term course will be affected. In view of the observations that UDCA can change the lipid profile and that patients with CF and liver disease are more likely to have essential fatty acid (EFA) deficiency, we elected to examine changes in the lipid profile and in the status of fat-soluble vitamins in response to UDCA. METHODS: Nineteen children with CF and liver dysfunction were recruited for a double-blind, crossover study of 1 year's duration, followed by treatment of the entire group. UDCA was administered at a dosage of 15 mg/kg per day, which, in the absence of a 50% decrease of alanine transaminase or aspartate transaminase or both within 2 months, was increased to 30 mg/kg per day. RESULTS: At entry, all patients had biochemical evidence of EFA deficiency. The lipid profiles during an average period of 25 months of follow-up showed a significant decrease in triglycerides (p <0.002), cholesterol (p <0.02), and total fatty acids (p <0.006). In addition, UDCA therapy led to an improvement in EFA status, as indicated by an increase (p <0.05) in the n-6 fatty acid concentration and a reduction (p <0.04) in the 20:3n-9/20:4n-6 fatty acid ratio. Although no change in vitamin E levels was observed, retinol metabolism was altered. There was an increase (p <0.02) in the unesterified retinol/retinol binding protein molar ratio in the absence of a difference in retinol binding protein concentration. Furthermore, retinyl esters, which normally account for less than 3% of circulating retinol, decreased (p <0.05) from 13.7% +/- 3.6% to 8.1% +/- 1.7%. CONCLUSIONS: This study confirms that UDCA alters lipoprotein metabolism and shows that it improves the EFA and retinol status of patients with CF and liver disease.

Imaging features of type 1 hereditary tyrosinemia: a review of 30 patients.

Hereditary tyrosinemia type 1, a common genetic disorder in the province of Quebec, is characterized by a deficiency of fumarylacetoacetate hydrolase. In this autosomal recessive disorder of tyrosine metabolism, the accumulation of succinylacetone leads to neurologic crises, acute and chronic liver failure, complex renal tubulopathy, rickets and a hemorrhagic syndrome. Liver trans- plantation has dramatically modified the spontaneous course of this lethal disease. The present paper describes the imaging features of tyrosinemia in 30 patients followed from 1980 to 1995 at Hôpital Sainte-Justine, Montreal, Canada.

Phenotypical and functional characterization of intrahepatic bile duct cells from common duct ligated mice.

Bile duct cells (BDCs), especially intrahepatic cholangiocytes, are primary targets of immune-related injury in such pathologic processes as liver graft rejection, liver graft-versus-host disease, and primary sclerosing cholangitis. Cholestasis and progressive loss of intrahepatic BDCs indicate chronicity in these diseases. The present investigation characterizes the acquisition of immune markers of intrahepatic BDCs isolated from mice after bile duct ligation. Purified BDCs from cholestatic C57BL/6 (H-2b) mice express not only the major histocompatibility complex (MHC) class I and class II antigens but also the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The expression of the MHC class II molecules on BDCs before and after interferon (IFN)-gamma exposure is also demonstrated by immunohistochemistry on the cultured cells. Cytotoxicity assays indicate the vulnerability of these cells as targets for immunologic injuries. Effector cells generated from B10.BR splenocytes (H-2k) against C57BL/6 splenocytes (H-2b) show comparable killing of BDCs and EL4 cells, the latter being a lymphoma line that was established from the C57BL/6N (H-2b) mouse. An immortalized mouse BDC line (IBDC) is included in this study to validate some of the findings from BDCs isolated from bile duct-ligated mice. We suggest that cholestatic BDCs express surface antigens different from those of normal epithelial cells that result in increased susceptibility to damage by immune mechanisms.

Tyrosinemia: the Quebec experience.

Tyrosinemia, a genetic disorder of the liver and kidneys, is caused by reduced activity of fumarylacetoacetate hydrolase (FAH), the final enzyme in the degradation of tyrosine. The consequent presence of succinylacetone in urine or blood is pathognomonic of tyrosinemia and is used as a confirmatory test in the Quebec neonaral screening program. Due to a complex founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia, particularly in the Saguenay-Lac Saint-Jean region (where the prevalence is 1 in 1850). Tyrosinemia has several different clinical presentations, ranging from acute liver failure with severe coagulopathy early in life, to slowly progressing cirrhosis with multiple nodules and variable renal dysfunction, to normal liver function with renal failure. Hepatocarcinoma has been found in approximately one third of cases. FAH complementary DNA has been cloned and mapped to chromosome 15q23-q25. The mutation observed in Quebec is a splice mutation at intron 12. This mutation is common and has been observed in other areas of the world as well, although more than 20 mutations causing tyrosinemia have now been described. Liver transplantation remains the definitive treatment. The author's team has carried out 28 liver transplantations (including 2 combined liver-kidney transplantations) in 25 children. The overall survival rate has been 92%; two children died as a result of primary nonfunction. The primary indications for transplantation were hepatic nodules (in 14 cases), neurological crises (6) and hepatic (3) or renal failure (2). An abnormal glomerular filtration rate (GFR) of less than 80 mL/min per 1.73 m2 was documented before transplantation in 54% of the cases. The rate normalized after liver transplantation in most patients, with rapid improvement in tubular function. However, patients with a severely low rate (less than 55 mL/min per 1.73 m2) before transplantation still had borderline renal function and poor growth after the transplantion, despite normal liver function. Therefore, for children with a consistently low GFR, careful consideration should be given to performing a combined liver-kidney transplantation, and a renal biopsy should form part of the pretransplantation evaluation.