Biomedical subjects
Publications and source records attributed to J D Perkins.
Obstruction of the IVC occurs in only 1-2% of patients after liver transplantation. The mortality of this complication can be as high as 66%. This case report describes the use of a Wallstent for an IVC obstruction that was unresponsive to conventional balloon angioplasty.
Focal rejection involves less than 20% of portal tracts in liver allograft biopsy results. The clinical significance of "focal" rejection on protocol liver biopsy results is unknown. The purpose of this study was to prospectively determine the incidence of clinically significant rejection in patients with focal rejection after orthotopic liver transplantation. Biopsy specimens from 165 consecutive transplantations in 149 patients were analyzed. After protocol biopsy specimens were obtained, patients with focal or mild rejection were observed. Fifty of 583 (8.6%) protocol biopsy results in 41 patients showed focal or mild rejection. None were treated on the basis of this histological finding. Six patients subsequently developed abnormal liver function tests and required treatment with additional immunosuppression. We conclude that focal or mild rejection is a relatively common finding on protocol liver biopsy results and only rarely progresses to a clinically significant problem. Patients with this finding can safely be observed without treatment.
The in vivo intestinal metabolism of the CYP3A probe midazolam to its principal metabolite, 1'-hydroxymidazolam, was investigated during surgery in 10 liver transplant recipients. After removal of the diseased liver, five subjects received 2 mg midazolam intraduodenally, and the other five received 1 mg midazolam intravenously. Simultaneous arterial and hepatic portal venous blood samples were collected during the anhepatic phase; collection of arterial samples continued after reperfusion of the donor liver. Midazolam, 1'-hydroxymidazolam, and 1'-hydroxymidazolam glucuronide were measured in plasma. A mass balance approach that considered the net change in midazolam (intravenously) or midazolam and 1'-hydroxymidazolam (intraduodenally) concentrations across the splanchnic vascular bed during the anhepatic phase was used to quantitate the intestinal extraction of midazolam after each route of administration. For the intraduodenal group, the mean fraction of the absorbed midazolam dose that was metabolized on transit through the intestinal mucosa was 0.43 +/- 0.18. For the intravenous group, the mean fraction of midazolam extracted from arterial blood and metabolized during each passage through the splanchnic vascular bed was 0.08 +/- 0.11. Although there was significant intersubject variability, the mean intravenous and intraduodenal extraction fractions were statistically different (p = 0.009). Collectively, these results show that the small intestine contributes significantly to the first-pass oxidative metabolism of midazolam catalyzed by mucosal CYP3A4 and suggest that significant first-pass metabolism may be a general phenomenon for all high-turnover CYP3A4 substrates.
OBJECTIVE: To determine in humans the relative roles of intestinal and hepatic metabolism in the oral first-pass elimination of a CYP3A substrate using midazolam as a model compound. METHODS: Midazolam was administered intravenously (1 mg) or orally (2 mg) to 20 healthy young subjects (10 men and 10 women) in a random fashion, and the disposition of the drug and its 1'-hydroxy metabolite were determined. In separate in vitro studies, the CYP3A-mediated formation of 1'-hydroxymidazolam by human hepatic and intestinal microsomes was investigated. RESULTS: No gender-related differences were noted in either the systemic (370 +/- 114 ml/min [mean +/- SD]) or oral (1413 +/- 807 ml/min) clearance values of midazolam. Despite complete oral absorption, measured oral bioavailability was on average about 50% less than that predicted on the assumption that only the liver contributed to first-pass metabolism. Pharmacokinetic estimation of the intestinal component indicated an extraction ratio (0.43 +/- 0.24) that was similar to that of the liver (0.44 +/- 0.14). 1'-Hydroxymidazolam was extensively but variably formed in vitro by both hepatic and intestinal microsomes and, although the intrinsic clearance (V(max)/Km) was higher in the liver preparations (540 +/- 747 versus 135 +/- 92 microliters/min/mg protein), this difference was not statistically significant. CONCLUSIONS: These results show that the small intestine can be a major site for presystemic, CYP3A-mediated metabolism after oral administration. Moreover, it appears that this represents a true first-pass effect. In addition, intestinal and hepatic metabolism may be important factors in interindividual variability in disposition after oral administration of midazolam and similar CYP3A substrates. Finally, intestinal localization of CYP3A may be significant in metabolism-based drug-drug interactions.
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To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.
Hepatitis C virus (HCV) infection may be associated with extrahepatic illness including renal disease. We investigated the clinical and virological characteristics of three patients who developed a mesangial proliferative and sclerosing glomerulopathy alone or in association with membranoproliferative glomerulonephritis after liver transplantation for end-stage liver disease secondary to HCV infection. Using polymerase chain reaction technology and the IgM RIBA assay, viral load, genotype and IgM antibody response to HCV in the setting of glomerulonephritis was evaluated. Within 1 year of transplantation, the patients showed decreased renal function, proteinuria and recurrent hepatitis C liver disease. Likewise, HCV viral load increased following transplantation, whereas the viral genotypes remained unchanged. Although the first patient presented with classic type II cryoglobulinemia in association with glomerulonephritis, the second patient developed an IgM directed specifically against the hepatitis C core antigen. The third patient developed a low-titered IgM directed against the hepatitis C core antigen with rheumatoid factor activity but without cryoglobulinemia. All of the patients show IgM in glomerular capillary walls by biopsy. One patient has shown a clinical response to interferon (IFN) alfa-2b therapy without evidence of hepatic allograft rejection. The second and third patients have not responded to IFN or developed hepatic rejection. This study suggests that HCV-associated glomerulonephritis may complicate liver transplantation in conjunction with the production of increased amounts of IgM of variable specificity. The posttransplant setting may provide a unique situation in which to investigate the specific requirements for the onset of renal disease.
The use of allograft biopsies to guide treatment after solid organ transplantation is a valuable tool in the detection and treatment of rejection. Prior development and use of the cystoscopically guided pancreatic allograft biopsy have allowed for more accurate and timely diagnosis of pancreatic allograft dysfunction, possibly contributing to our 1-year pancreas graft, renal allograft and patient survival rates of 87.1%, 88.5% and 96.8%, respectively. We reviewed our experience, examining efficacy and complication rates of pancreas and kidney biopsies in 31 cadaveric pancreas or combined kidney and pancreas transplants performed between June 1990 and February 1992 with at least 1 year of followup. There were 94 pancreas, 54 kidney and 53 duodenal mucosal biopsies in 29 evaluable patients. This biopsy technique uses a 24.5F side-viewing nephroscope to view the cystoduodenostomy, with the duodenum acting as a portal for biopsy needles into the pancreas. Pancreatic tissue is obtained with either an 18 gauge, 500 mm. Menghini aspiration/core needle or an 18 gauge, 500 mm. Roth core needle. Percutaneous renal allograft biopsies are performed independently or simultaneously with the pancreas biopsies using a 16 gauge spring loaded needle. Pancreas biopsies were prompted by clinical indications of rejection (decreased urinary amylase, increased serum amylase or increased serum creatinine) or by protocol (10, 21 and 40 days postoperatively). Among the biopsies 30% were required by protocol, of which 10 (36%) revealed abnormal pathological findings and 5 (18%) showed evidence of occult cellular rejection. Renal biopsies demonstrated rejection in 69% of the cases. Of simultaneous pancreas/kidney biopsies 33% revealed concomitant rejection. A total of 88 Menghini needles with 170 passes was used in 73 biopsy attempts, yielding 126 tissue cores with a 16% complication rate. A total of 41 Roth needles was used with 73 passes in 34 biopsy attempts, yielding 55 tissue cores with a complication rate of 21%. Complications included self-limited bleeding from the biopsy site in 13% of the cases, bleeding requiring clot evacuation and fulguration in 1% and asymptomatic hyperamylasemia in 12%. Renal biopsy complications included 1 arteriovenous fistula (2%). We conclude that ultrasound and cystoscopically guided pancreatic allograft biopsy and percutaneous renal allograft biopsies are safe and essential methods of obtaining tissue for histological diagnosis without serious sequelae. The Menghini and Roth needles in cystoscopically guided pancreatic allograft biopsy have similar yield and complication rates in obtaining pancreatic tissue, although they require different performance techniques. In some cases both needles are necessary and are complementary in obtaining adequate tissue.(ABSTRACT TRUNCATED AT 400 WORDS)
We report a prospective clinical and virological study of 18 patients undergoing orthotopic liver transplantation, selected because of hepatitis C virus (HCV) RNA positivity before transplantation. Nine of the 18 patients (50%) developed chronic active hepatitis (CAH) in liver allografts during the first year posttransplantation; hepatitis was first observed between 6 and 25 weeks posttransplantation. HCV viremia was measured for all patients before transplantation and on posttransplantation days 3, 7, and 14, and months 1, 6, 12, and 24 to 41, by quantitative competitive RNA polymerase chain reaction (QC-PCR). HCV RNA levels on posttransplantation days 3, 7, and 14 were significantly higher among patients who subsequently developed CAH versus those who did not (P < .02 by t-test and Mann-Whitney test on all three dates). However, HCV RNA levels in sera obtained at 1, 6, and 12 months posttransplantation did not correlate with CAH at 1 year or with HCV genotype determined in posttransplantation sera. At least two serial liver biopsy specimens from each patient were stained for HCV nonstructural 4 (NS4) antigen by immunohistochemistry. The intensity of cytoplasmic staining of NS4 antigen was significantly higher for specimens with CAH versus those without CAH (P = .028 by chi 2). Three patients developed bridging fibrosis in liver allografts during the first year after transplantation; all three patients had intense (3+) immunostaining for NS4 antigen, and the infecting genotypes were 1a, 1b, and 1a plus 1b, respectively. In summary, the 18 patients all developed high-titer viremia by 1 month after liver transplantation, whereas CAH developed in 50% of allografts during the first year after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)
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Immunosuppression therapy with cyclosporine is often hampered by significant interindividual variability in the metabolic clearance of the drug. It has been suggested that much of the variability in cyclosporine clearance is due to differences in the cytochrome P450 3A4 (CYP3A4) content in the liver and intestinal mucosa. A study was conducted in liver transplant recipients to characterize hepatic CYP3A variability during the first 10 days after surgery. The formation of 1'-hydroxymidazolam (1'-OH MDZ) was followed in the plasma after i.v. midazolam (MDZ) administration to 21 multiple-organ donors and to recipients of 10 of the 21 donor livers. Liver biopsy tissue was obtained from donors and recipients after the in vivo pharmacokinetic test. For liver donors, the plasma 1'-OH MDZ/MDZ concentration ratio 30 min after the i.v. MDZ dose was well correlated with the hepatic CYP3A4 content (r = .87, P < .001). Much of the variability in the two parameters was attributed to the administration of enzyme-inducing drugs before organ procurement. The mean hepatic CYP3A4 content and plasma 1'-OH MDZ/MDZ concentration ratio in six inducer-treated donors was 4.7-fold and 2.3-fold higher than the respective mean value for all other donors. The hepatic CYP3A4 content and plasma 1'-OH MDZ/MDZ ratio for liver recipients, studied on postoperative day 10, was negatively correlated with the respective parameter measured in donors on day 0 (r = -0.60 for CYP3A4 and r = -0.79 for 1'-OH MDZ/MDZ; P < .05 and P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)
Complete XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 are presented, along with a comparison of the physical map of MG1655 with that of five other K-12 strains. We have mapped 35 XbaI cleavage sites generating 35 fragments ranging in size from 8 kb to 432 kb using methods similar to those used previously for the NotI and SfiI maps of MG1655. The applicability of the MG1655 map to other strains of E. coli K-12 was assessed by comparing the NotI, SfiI and XbaI digestion patterns of EMG2, W1485, W3110, AB1157 and MC4100 with those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the differences between the strains on the basis of simple insertions, deletions and, in one case, an inversion. Insertions and deletions ranging in size from 1 kb to 86 kb are suggested by this model. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains. The various features localized in these strains were used to place 9 of the 17 BlnI fragments on the E. coli physical map. The remaining fragments were placed by hybridization experiments similar to those used for the NotI, SfiI and XbaI maps. In this way, the complete BlnI map was constructed. The cleavage sites for XbaI and BlnI were assigned coordinates based on EcoMap6 developed by Rudd et al. The XbaI and BlnI maps of MG1655 presented here, when combined with the NotI (22 sites) and SfiI (31 sites) maps of MG1655 previously published, bring the total number of mapped rare restriction sites in MG1655 to 105. The strain comparison analysis shows that this map is readily adaptable for use with other K-12 strains.
A means to monitor intestinal allografts will be crucial for the future success of small bowel transplantation. We have previously demonstrated the ability of high-frequency ultrasound (US) to diagnose porcine intestinal ischemia in vitro. The aim of this study was to compare the histologic appearance of normal porcine small bowel versus bowel undergoing acute rejection, using 8.5-MHz US images. We allowed porcine heterotopic small bowel allograft transplants to reject and then removed, at scheduled intervals from postoperative day 0 to 12, specimens of both the transplanted bowel and the native bowel. We examined the tissues in vitro with an 8.5-MHz linear array US system then studied them histologically. Histologically, the earliest changes of rejection occurred at days 4 to 5, with mild submucosal edema, endotheliitis, and vasculitis affecting the small vessels; the mucosa remained normal. By days 7 to 8, the submucosal endotheliitis became more prominent, with focal thrombosed small vessels; the mucosa now appeared abnormal with flattened villi, erosions, and necrosis. By days 10 to 12, marked submucosal edema, vasculitis, endotheliitis, and necrotic mucosa were present. Ultrasonically, normal intestinal wall has five wall layers, corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. The US criteria for abnormality were loss of folds, decreased numbers of echo layers, discontinuity of layers, and a homogeneous appearance. Using these US criteria, blinded observers differentiated normal from abnormal bowel wall after transplantation with a sensitivity of 84% and a specificity of 81%. Most of the errors occurred with the day 4 and 5 specimens, which appeared nearly normal on US.(ABSTRACT TRUNCATED AT 250 WORDS)
Although surgical proficiency is essential to the immediate outcome of transplantation, long-term success depends upon how adequately the transplantation recipient is managed. Immunosuppression, the most critical aspect of after care, is subject to wide variation. In January 1990, a survey was sent to the directors of all transplant programs in the United States performing one or more kidney, heart, liver, heart-lung, or pancreas transplant in 1988. Detailed data were obtained on both the drugs and methods used for induction and maintenance immunosuppression, as well as the treatment of rejection. Each program director was asked to rank each immunosuppressive approach according to its perceived impact on patient outcomes. Over 85% of all eligible program directors completed the survey. There is no evidence of survey respondent bias. The use of polyclonal and monoclonal agents for induction immunosuppression was favored most by pancreas program directors (72-76%). These agents were least preferred by liver transplant programs (35-37%). About half of kidney, heart, and heart-lung program directors preferred these agents. Triple-drug therapy consisting of CsA, PRED, and AZA was considered the most preferable maintenance protocol for all transplants (i.e., kidney, 89%; heart, 94%; liver, 88%; heart-lung, 86%; pancreas, 96%). Either i.v. steroids or OKT3 were regarded as the preferred approaches for the treatment of acute or resistant rejection. Finally, the acceptability of outpatient treatment of rejection varied by transplant type (i.e., kidney, 9%; heart, 58%; liver, 5%; heart-lung, 29%; pancreas, 8%). Although there are similarities in the ratings of various aspects of immunosuppressive therapy, there are important differences. This information is critical to anticipate the implications of new immunosuppressive agents and to evaluate changes in the use of existing drugs and therapeutic approaches.
VCAM-1, a leukocyte adhesion molecule expressed by cytokine-activated endothelial cells in culture, may mediate mononuclear leukocyte infiltration in vessels and interstitium in solid organ allograft rejection. Using the avidin-biotin immunoperoxidase technique and two different antibodies--4B9, a murine monoclonal antibody, and rabbit polyclonal antisera to recombinant human VCAM (rVCAM Ab), which work in methacarn-fixed tissues--we studied the expression of this molecule in biopsies of transplanted liver and pancreas with and without features of rejection as well as nontransplant control tissues. The rVCAM Ab, but not 4B9, showed a population of reactive endothelial cells limited to sites of prominent subendothelial leukocytic cell infiltration in arteries and veins in rejecting allografts. VCAM-1 expression by sinusoidal endothelium in rejecting liver allografts was also observed. In addition, a population of cells (DC) with dendritic morphology was identified by rVCAM Ab within sites of lymphoid cell aggregations in both liver and pancreas allografts. Further evidence that these cells represent true DC was obtained by identification of VCAM-1 positive morphologically similar cells in both germinal centers and interfollicular areas of reactive lymph nodes; and by similar staining of these cells in allograft organs by a monoclonal antibody to nerve growth factor receptor, previously shown to recognize DC. DCs were generally not seen in normal control organs or portions of allografts uninvolved by lymphoid aggregates. This study provides evidence that 1) endothelial cell expression of VCAM-1 may be important in transplant rejection, 2) different epitopes of VCAM-1 may be preserved in tissue sections and recognized by different antibodies, and 3) there is probably a population of VCAM-1 expressing DC that participates in the cellular rejection progress.
An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.
To evaluate the use of ultrasound (US) to detect intestinal wall ischemia, we isolated segments of jejunum on single vascular pedicles in five piglets. We sequentially clamped these segments in intervals of 0 to 6 h, reperfused them for 24 h, and then examined them in vitro histologically and with an 8.5 MHz US scan. All segments were grossly viable except those with 6 h of ischemia. Histologically, mild submucosal edema developed after 1 to 2 h of ischemia; after 3 to 4 h, mucosal necrosis, loss of folds, worsening submucosal edema, and prominent neutrophilic infiltration occurred; after 5 to 6 h, severe mucosal necrosis with hemorrhage and submucosal edema was present. Ultrasonically, we saw five wall layers in the control group corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. After mild (1 to 2 h) ischemia, all layers were present except for a discontinuity in layer 3. After moderate (3 to 4 h) ischemia, the five layers persisted but with a markedly thickened submucosal layer, reduced echogenicity, and flattened mucosal folds. With severe (5 to 6 h) ischemia, we observed a loss of all normal layers with no discernible architecture. Using these US criteria, blinded observers were able to differentiate normal/mild from moderate/severe ischemia with a sensitivity and specificity of 100%. These data suggest that US can differentiate, in vitro, normal from moderate and severe degrees of intestinal wall ischemia that correlates well with the histological appearance. Endoscopic US or surgically implantable US probes can potentially help diagnose clinical intestinal wall ischemia.
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