Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver.

The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress.

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.

Right ventricular performance using myocardial deformation imaging in infants with bronchopulmonary dysplasia.

OBJECTIVE: Right ventricular (RV) performance among infants with bronchopulmonary dysplasia (BPD) remains poorly understood. We tested the hypothesis that myocardial deformation imaging (MDI) strain and strain rate would allow for differentiation between infants with severe and milder forms of BPD, independent of tissue Doppler imaging (TDI) and superior to conventional echocardiographic measurements. STUDY DESIGN: Infants with various severities of BPD (11 with none or mild, 13 with moderate and 10 with severe) underwent conventional echocardiography, TDI and MDI assessments at >36 weeks of corrected gestational age. BPD severity grading was determined according to the National Institutes of Child Health and Disease workshop rating scale by physicians blinded to the echocardiogram results. Group data were compared with one-way analysis of variance or Kruskal-Wallis tests, with post hoc multiple comparisons. RESULTS: No differences in traditional echocardiographic parameters or TDI among the three BPD severity groups were observed; none of the infants had evidence of pulmonary hypertension. Using MDI, infants with severe BPD had lower peak global systolic strain than did infants with moderate BPD (P<0.01) or mild/none BPD (P<0.01). Early and late diastolic strain rate measurements were similar across the three groups. CONCLUSIONS: Among infants with severe forms of BPD, evidence of abnormal RV systolic function was detected with MDI, but not traditional echocardiographic or TDI measurements. Infants with severe forms of BPD may represent a particularly high-risk subgroup for decreased RV performance warranting cardiac surveillance. MDI should be considered as a method to quantitate RV function in this population.

Preparedness of pediatric residents for fellowship: a survey of US neonatal-perinatal fellowship program directors.

OBJECTIVE: To evaluate the preparedness of pediatric residents entering accredited neonatal-perinatal medicine (NPM) fellowships in the United States. STUDY DESIGN: A multi-domain, validated survey was distributed to Program Directors (PDs) of US NPM fellowship programs. The 47-item survey explored 5 domains: professionalism, independent practice, psychomotor ability, clinical evaluation, and academia. A systematic, qualitative analysis on free-text comments was also performed. RESULTS: Sixty-one PDs completed the survey, for a response rate of 62% (61/98). For entering fellows, PDs assessed performance in professionalism positively, including 76% as communicating effectively with parents and 90% treating residents/house-staff with respect. In contrast, most PDs rated performance in psychomotor abilities negatively, including 59% and 79% as deficient in bag-and-mask ventilation and neonatal endotracheal intubation, respectively. Although 62% of PDs assessed entering fellows positively for genuine interest in academic projects, fewer than 10% responded positively that entering fellows understood research protocol design, basic statistics, or were capable of writing a cohesive manuscript well. Thematic clustering of qualitative data revealed deficits in psychomotor ability and academia/scholarship. CONCLUSIONS: On the basis of the perspective of front line educators, graduating pediatric residents are underprepared for subspecialty fellowship training in NPM. To provide the best preparation for pediatric graduates who pursue advanced training, changes to residency education to address deficiencies in these important competencies are warranted.

Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein.

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A. The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da). Complete native data have been collected to 2.5 A resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A. The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution. Molecular replacement studies of both complexes are underway.

Early versus delayed umbilical cord clamping in infants with congenital heart disease: a pilot, randomized, controlled trial.

OBJECTIVE: Delayed umbilical cord clamping (DCC) at birth may provide a better neonatal health status than early umbilical cord clamping (ECC). However, the safety and feasibility of DCC in infants with congenital heart disease (CHD) have not been tested. This was a pilot, randomized, controlled trial to establish the safety and feasibility of DCC in neonates with CHD. STUDY DESIGN: Pregnant women admitted >37 weeks gestational age with prenatal diagnosis of critical CHD were enrolled and randomized to ECC or DCC. For ECC, the umbilical cord was clamped <10 s after birth; for DCC, the cord was clamped ~120 s after delivery. RESULTS: Thirty infants were randomized at birth. No differences between the DCC and ECC groups were observed in gestational age at birth or time of surgery. No differences were observed across all safety measures, although a trend for higher peak serum bilirubin levels (9.2±2.2 vs 7.3±3.2 mg dl(-1), P=0.08) in the DCC group than in the ECC group was noted. Although similar at later time points, hematocrits were higher in the DCC than in the ECC infants during the first 72 h of life. The proportion of infants not receiving blood transfusions throughout hospitalization was higher in the DCC than in the ECC infants (43 vs 7%, log-rank test P=0.02). CONCLUSION: DCC in infants with critical CHD appears both safe and feasible, with fewer infants exposed to red blood cell transfusions than with ECC. A more comprehensive appraisal of this practice is warranted.

In vitro and in vivo effects of recombinant human interleukin-2 in naive miniature swine.

Recent data in mice have shown that early administration of recombinant human interleukin-2 (rIL-2) provides significant protection from lethal graft-versus-host disease. Because of the potential clinical importance of these findings, it will be important to assess the effectiveness of this therapy in a large animal preclinical bone marrow transplantation model. We report here our initial studies of the in vitro and in vivo effects of rIL-2 in miniature swine. In vitro 4-day cultures of pig peripheral blood lymphocytes (PBL) in complete medium containing rIL-2 at 1,000 U/ml resulted in optimal proliferation and generation of lymphokine-activated killer (LAK) cells. A pig-mouse hybridoma cell line was found to be highly sensitive as a LAK cell target. Two naive pigs received 20,000 U/kg and 2 pigs received 100,000 U/kg of rIL-2 intravenously twice a day for 4 days. No clinical symptoms were seen during or after administration at the lower dose while both high dose-treated animals showed generalized erythema from days 2 to 4, and one showed mild diarrhea during this period. The disappearance of IL-2 activity from the serum showed two components: (1) an initial fast component with a half-time of approximately 10 min and (2) a slow component with a half-time of approximately 60 min. LAK cell precursors disappeared from the peripheral circulation by 6 min after rIL-2 administration and began to recover by 6 h in the low dose recipients and only after 12 h in the high dose recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of hydrazine metabolites formed from isoniazid.

Studies in rats indicate that the metabolic activation of acetylhydrazine, a metabolite of isoniazid, is a critical determinant of the hepatotoxicity of isoniazid. As demonstrated in that model, the formation of 14CO2 after the administration of 14C-acetylisoniazid reflects the activity of the toxic pathway. A similar approach in man should make it possible to demonstrate the presence and to assess the quantitative importance of this toxifying pathway, and thus to evaluate its role in the pathogenesis of isoniazid hepatitis. We gave 300 mg isoniazid together with 10 microCi 14C-acetylisoniazid (12 mg) to 17 healthy subjects and determined the time course of the plasma concentrations of isoniazid, acetylisoniazid, acetylhydrazine, and diacetylhydrazine and of the exhalation of 14CO2. The time course of 14CO2 in breath closely paralleled the plasma concentration-time curve of acetylhydrazine but not those of acetylisoniazid or diacetylhydrazine, indicating that the 14CO2 originated directly from the metabolism of acetylhydrazine. The cumulative exhalation of 14CO2 increased with decreasing rate of acetylation of isoniazid, such that slow acetylators generated more 14CO2 than rapid acetylators. Simulation studies demonstrated that even if the data are corrected for the different formation of acetylisoniazid from isoniazid in slow and rapid acetylators, the slow acetylators still generated more 14CO2. The data therefore indicate that a substantial fraction of the acetylhydrazine formed from isoniazid passes through a pathway that has been shown in animals to generate highly reactive and hepatotoxic intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlations and apparent contradictions in assessment of oxidant stress status in vivo.

Oxidative modifications of biological molecules are essential, but uncontrolled or excessive oxidative activities appear to contribute to many disease states. The mechanisms through which excess oxidant activities cause injury have been studied most extensively for acute responses, particularly for drug-induced tissue damage and cell death, but substantial evidence suggests that chronically elevated oxidative activity may contribute to the development of diseases such as cancer. It is important that the correlation between oxidant stress status and cancer risk be examined directly in humans. A number of methods have been developed for assessing oxidant activities by measuring oxidized products in biological systems, but cross-comparison studies of these different methods are needed. In studies of mechanisms of acute hepatotoxicity, assessments of oxidant stress responses by different analytical methods often have provided data that appear at first glance to be contradictory. Marked oxidant stress responses may be indicated by one or more methods of analysis despite the lack of detectable change in other parameters, whereas in a second experimental model the responses may be reversed. These observations emphasize the need to integrate different analytical approaches into the assessment of oxidant activity in vivo and illustrate the importance for developing a better understanding of the chemical and physiological mechanisms through which the analytical methodologies are related.

Methods for determination of lipid peroxidation in biological samples.

Interest in the pathological consequences of lipid peroxidation has led to the development of a number of analytical approaches to the quantitation of products. However, the various analytical methodologies employed often do not measure the same chemical classes of products, and apparent discrepencies have been observed, particularly in studies of lipid peroxidation in biological systems. This review provides a brief discussion of some of the strengths and weakness of methods currently used for the determination of lipid peroxidation in biological tissues.

Hyperoxic lung injury in Fischer-344 and Sprague-Dawley rats in vivo.

Supplemental oxygen remains an important therapy for pulmonary insufficiency, despite the potential adverse effects of hyperoxic exposures. Recently, He et al. reported that hyperoxic ventilation more readily damaged isolated perfused lungs from Fischer-344 rats than from Sprague-Dawley rats (Am. J. Physiol. 259:L451), which correlates with the previously reported strain differences in hepatic responses to diquat-induced oxidant stress in vivo (J. Pharmacol. Exp. Ther. 235:172). We therefore examined the differences in hyperoxic lung injury in Fischer-344 and Sprague-Dawley rats in vivo. Adult male rats were exposed to > 95% O2 and were sacrificed after 24, 48, or 60 h. Control animals were maintained in room air. Dramatically greater increases in pleural effusions and bronchoalveolar lavage protein concentrations in response to hyperoxia were observed in the Fischer-344 rats than in the Sprague-Dawley rats (p < .05 at both 48 and 60 h for both measurements). Additionally, the glutathione concentrations in alveolar lining fluid decreased from 800 microM to 115 microM in Fischer-344 rats after 60 h of > 95% O2, but did not change in Sprague-Dawley rats. We conclude that the greater susceptibility of Fischer-344 than of Sprague-Dawley rats to hyperoxic lung injury in vitro reported previously also is observed in vivo and that this strain difference offers unique opportunities to study mechanisms of hyperoxic lung injury.

Oxidative modifications of apoB-100 by exposure of low density lipoproteins to HOCL in vitro.

Although the products of oxidation of the lipid components of LDL have been studied extensively, much less is known about the specific products of oxidative modification of the apoprotein. We reacted native LDL and LDL that had been treated with HOCl with 2,4-dinitrophenylhydrazine (DNPH), delipidated and trypsinized the protein, and analyzed the products by HPLC. Although tryptic digests of native LDL and LDL oxidized by limited quantities of HOCl showed similar patterns by HPLC with detection at 220 nm, oxidized LDL showed several discrete peaks at 365 nm, which is characteristic of hydrazones formed with aldehydes and ketones, commonly termed protein carbonyls. Native LDl showed no peaks in the chromatograms at 365 nm. Peptides absorbing at 365 nm were isolated by HPLC and characterized. In most cases, the probable sites of modification on the peptides could be implied by failure of an anticipated amino acid to appear in the expected sequence. Of the 14 peptides isolated and characterized to date, eight peptides contained Cys residues. In other peptides, Lys, Trp, and Met were identified as amino acid residues apparently modified by HOCl treatment of LDL. Thirteen of the peptides identified are from trypsin-releasable peptides located on the surface of unoxidized native LDL. Our studies suggest a selective process of modification of apoB-100 by HOCl and the approaches used in the present studies should be useful for the characterization of the mechanisms of oxidation of this and other proteins.

Oxidation of bovine beta-casein by hypochlorite.

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O(2) for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat beta-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine beta-casein by HOCl. Following exposure to HOCl at 4 degrees C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177-183 of bovine beta-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH+ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH+ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y(193) within the tryptic peptide constituted by amino acids 184-202, and the corresponding chymotryptic cleavage side product, 191-202. Exposure of beta-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine beta-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models.

Toxicity of oxidized low density lipoproteins for vascular smooth muscle cells and partial protection by antioxidants.

Oxidized low density lipoprotein (oxLDL) is known to be toxic to a variety of cell types, but relatively little is known about the toxic effects of oxLDL on vascular smooth muscle cells (SMC). We found that LDL oxidized by incubation with 5 microM cupric ions was toxic to cultured porcine SMC when administered at concentrations of 25 micrograms protein/ml and higher. The toxicity was demonstrated whether cells were proliferating or not, and was more evident in the presence of 0.4% lipoprotein-deficient serum than in 10%. Because of recent evidence that 7-ketocholesterol and 7-hydroxycholesterol are toxic species in copper-oxidized LDL, inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase was hypothesized as a mechanism of toxicity. However, mevalonic acid, the product of this enzyme, failed to protect against the toxicity of either oxLDL or the pure oxysterols. Alpha-tocopherol, alpha-tocopherol acetate, probucol, butylated hydroxytoluene, and deferoxamine provided partial protection to SMC exposed to oxLDL. These results suggested a toxic role for newly initiated lipid peroxidation, either in cells or in media oxLDL. Cellular lipid peroxidation appeared more likely, since no further oxidation of media oxLDL was demonstrated in the presence or absence of antioxidants. Overall, the results suggest that toxicity of copper-oxidized LDL for SMC is multifactorial and differs from the previously described toxicity of iron-oxidized LDL for fibroblasts.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Urgent revascularization of liver allografts after early hepatic artery thrombosis.

Between April 1993 and May 1995, 17 adult orthotopic liver transplant recipients were found to have early hepatic artery thrombosis (HAT) after a median of 7 postoperative days (mean, 11). The HAT was diagnosed in all cases by duplex ultrasound. Thrombectomy was performed with urgent revascularization (UR), using an interposition arterial graft procured from the cadaveric liver donor, and arterial patency was verified with intraoperative angiography. In seven cases, intra-arterial urokinase was administered after the thrombectomy. Fifteen (88%) of the livers remained arterialized throughout the follow-up period (median, 15 months); the remaining two patients developed recurrent HAT after 6 and 8 months. Although there was a high rate of subsequent complications, 11 (65%) of the patients are alive without retransplantation, with a mean follow-up of 17 months. Despite having a patent hepatic artery, the remaining six patients (35%) died from infectious complications that usually were present before the UR. Thus, UR effectively restored arterial inflow in 88% of the patients with early HAT. The ultimate outcome was determined mainly by the presence of intra-abdominal complications at the time of UR. In conclusion, UR, rather than retransplantation, should be considered the prime treatment option for patients who develop early posttransplant HAT.

Bone marrow transplantation in miniature swine: IV. Development of myeloablative regimens that allow engraftment across major histocompatibility barriers.

Studies of the myeloablative regimens capable of permitting successful BMT across MHC barriers in miniature swine have been performed. To minimize graft-versus-host disease (GVHD), engraftment was studied in the F1-->P combination (i.e., MHC homozygous ["parental"] swine receiving bone marrow from one-haplotype matched MHC heterozygous ["F1"] donors). Animals given total body irradiation (TBI) up to 1100 cGy, 10 cGy/min, in a single dose failed to engraft. Increasing the dose rate led to unacceptable extramedullary toxicity without improving engraftment. Eleven different fractionated TBI regimens were tested in this F1-->parent model. At all of the dose rates tested, a total dose of less than 1000 cGy was insufficient for engraftment, and a total dose of 1400 cGy led to unacceptable toxicity. Between these extremes, a window was defined in which engraftment could be obtained without unacceptable extramedullary toxicity utilizing 2 equally divided fractions of TBI delivered 24 hr apart. The addition of 50 mg/kg cyclophosphamide i.v. to fractionated TBI (1150 cGy total dose [500 + 650]) also permitted engraftment, with decreased incidence of interstitial pneumonitis as compared to fractionated TBI (1300 cGy total dose [650 x 2]). Both of these regimens were also confirmed to permit engraftment between heterozygous donors and recipients sharing a single common haplotype ("F1-->F1"). The regimen of 1300 cGy (650 x 2) also permitted engraftment in completely MHC mismatched BMT, but with subsequent death from GVHD. These studies of the myeloablative regimens permitting engraftment across defined MHC barriers in miniature swine provide a basis for further studies of allogenic BMT and GVHD in this large animal preclinical model.

Successful induction of long-term specific tolerance to fully allogeneic renal allografts in miniature swine.

Major histocompatibility complex class II matching is of overwhelming importance for achieving tolerance to kidney transplants (KTX) in miniature swine. When class II antigens are matched, long-term specific tolerance across complete MHC class I antigen barriers can uniformly be induced by a 12-day perioperative course of cyclosporine. This same regimen is ineffective in fully MHC-mismatched combinations. We hypothesized that initial induction of tolerance to kidney donor class II antigens by bone marrow transplantation might allow tolerance to be induced to a subsequent fully allogeneic KTX in combination with CsA therapy. We report here the results of such fully allogeneic KTX performed in 4 recipients of prior single-haplotype class II-mismatched BMT. All animals received KTX from donors class II matched to the BMT donor and received a 12-day course of intravenous CsA (10 mg/kg/day). All four animals have maintained normal serum creatinine values (less than 2.0 mg/dl) for greater than 200 days posttransplant. Specific hyporesponsiveness to both BMT and KTX donor-type MHC antigens was found in mixed lymphocyte culture and cell-mediated lympholysis assays. Compared with third-party grafts, significantly prolonged survival of BMT donor-specific (P = 0.031) and KTX donor-specific (P = 0.031) skin grafts was observed. These results demonstrate that induction of tolerance to class II antigens by BMT allows a short course of CsA to induce specific tolerance to fully allogeneic renal allografts.

The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells.

Bone marrow transplantation has been shown to induce donor-specific tolerance in rodent models. This approach could potentially be applied to xenotransplantation across discordant species barriers. To evaluate host factors resisting hematopoietic cell engraftment, we have developed two model systems utilizing the combination of swine into severe combined immunodeficient (SCID) mice. SCID mice lack functional B and T lymphocytes, and can therefore be used to evaluate nonimmune factors resisting marrow engraftment, and for adoptive transfer studies to test the role of immune cells and antibodies. First we transplanted swine bone marrow cells into SCID mice conditioned with whole-body irradiation (4 Gy). For nine weeks following the intravenous administration of 10(8) swine bone marrow cells, up to 3.8% of peripheral blood leukocytes were of swine origin, as determined by flow cytometry (FCM). These cells were all of the myeloid lineage. Swine IgG was also detectable in the serum for up to 14 weeks. The bone marrow of the reconstituted mice contained low percentages of swine myeloid cells, and swine myeloid progenitors could be detected for up to 20 weeks after bone marrow transplantation. In a second model, we grafted thymus and liver tissue from 45-69-day-old swine fetuses under the kidney capsule of 4 Gy-irradiated SCID mice. A suspension containing 10(8) swine fetal liver cells (FLC) was also administered i.p. Long-term repopulation with swine T cells was observed, with up to 1.5% swine T cells detected in the WBC, peritoneum, and spleen for at least 5.5 months postgrafting. These T cells expressed either CD4 or CD8, whereas up to 17.6% of cells in the thymic grafts expressed both CD4 and CD8. The i.p. FLC suspension was required for optimal long-term graft maintenance. Our studies show that (1) low level myeloid and B lymphocyte reconstitution can be achieved by transferring adult swine BMC to irradiated SCID recipients; (2) swine myeloid progenitors were detectable long-term in BMC of these mice, suggesting that stem cell engraftment was achieved; and (3) T cell reconstitution of SCID mice by swine progenitors requires cotransplantation of a swine stromal environment, as is provided by fetal swine thymus/liver grafts. We conclude that nonimmune factors such as those provided by species-specific stromal environments are important for reconstitution of some lineages by discordant hematopoietic stem cells.

Improved human islet isolation by a tube method for collagenase infusion.

BACKGROUND: Collagenase infusion into the pancreatic duct is an essential step in human islet isolation. We developed a new method for ductal canulation and collagenese infusion. METHODS: A total of 53 pancreata were divided into two groups: group 1 (n=23), the new tube method, and group 2 (n=30), the standard angiocatheter method. In group 1, a polyethylene tube was inserted into the duct and pushed to the tail. The tail was first expanded, followed by expansion of the body and then the head, by pulling out the tube. RESULTS: Total islet number and number/g pancreas (mean+/-SE) were significantly higher in group 1 (481,123+/-43,218 and 8,010+/-722) (mean+/-SE) than in group 2 (300,974+/-35,122 and 5,090+/-515, P<0.01). Total islet equivalent number and islet equivalent number per gram pancreas were also significantly higher in group 1 (319,176+/-39,354 and 5,455+/-652) than in group 2 (202,022+/-23,331 and 3,722+/-468, P<0.04). Islet purity and fragmentation showed no differences between the groups. CONCLUSIONS: The tube method improved islet yields. We recommended this method for human islet isolation.