Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well-preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Development of the isolated dual perfused rat liver model as an improved reperfusion model for transplantation research.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Cytogenetic analysis of murine embryo-derived tumors.

The possible relationship among malignancy, differentiation, and chromosomal constitution of primary embryo-derived tumors was studied. Tumors were induced by transplanting 7-day-old mouse embryos under the kidney capsule of syngeneic BALB/c recipients. Transplantation of 101 embryos resulted in 18 tumor-bearing mice: 36 teratocarcinomas; 18 teratomas; and 27 yolk sac tumors. Some of the yolk sac tumors proved to be retransplantable for several generations. Cytogenetic investigation of the primary embryo-derived tumors revealed that the majority of teratocarcinomas (82%) were chromosomally normal, whereas almost all (83%) karyotyped teratomas and yolk sac tumors had a highly abnormal chromosomal constitution. Most common aberrations were polyploidy; overrepresentation of chromosome 1, 6, 15, or 19; and an underrepresentation of chromosome 2, 4, 14, or a sex chromosome.

Chromosomal changes in mature residual teratomas following polychemotherapy.

A cytogenetic analysis of 13 mature residual teratomas following chemotherapy revealed modal chromosome numbers ranging from 52 to 85, in agreement with the flow cytometric determination of the DNA content of the tumors. At least one copy of an i(12p) was present in 12 tumors. One tumor, however, lacked that marker. The comparison between the chromosomal abnormalities found in mature residual teratomas following chemotherapy and those from primary testicular nonseminomas suggests that residual teratomas result from selection of clones from the primary tumor with a less abnormal karyotype.

Hypothermic machine perfusion of the liver and the critical balance between perfusion pressures and endothelial injury.

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.

Cytogenetic study of a combined germ cell tumor of the testis.

The cytogenetic findings in both components of a combined germ cell tumor of the testis are described. The only structural chromosomal abnormality in common was an i(12p).

Cytogenetics of carcinoma in situ of the testis.

Carcinoma in situ (CIS) of the testis is the precursor lesion of most testicular germ cell tumors (TGCTs). Karyotyping of CIS is important for a better understanding of the pathogenesis of TGCTs and the progression to invasive cancer. We karyotyped three cases of CIS. All three cases showed a numerical abnormal chromosomal pattern. In one case, two copies of the germ cell tumor marker i(12p) were found.

A residual mature teratoma with a more balanced karyotype than the primary testicular nonseminoma?

We have been able to study the karyotypes and measure the DNA content in both the primary nonseminomatous germ cell tumor of the testes and the residual mature teratoma after chemotherapy of the same patient. Based on these and other studies, the mechanism of therapy-related differentiation in germ cell tumors of the testes is discussed.

Karyotyping and DNA flow cytometry of an orchidoblastoma.

The first karyotype of an orchidoblastoma is described. The most striking finding is the absence of the i(12p) marker chromosome, considered specific for testicular germ cell tumors of adults. Differences between infantile and adult testicular germ cell tumors are discussed, as are features that infantile testicular germ cell tumors have in common with extragonadal germ cell tumors.

i(12p)-negative testicular germ cell tumors. A different group?

Cytogenetic analysis was performed of three seminomas, two primary nonseminomas, and two mature residual teratomas following chemotherapy, all lacking i(12p). Testicular germ cell tumors without an i(12p) may represent a subgroup of germ cell tumors, also in their clinical course, compared with those having i(12p).

Cytogenetics of thyroid follicular adenomas.

We present the results of a cytogenetic study of three cases of follicular adenoma of the thyroid. All three cases had a numerical strongly abnormal karyotype with most abnormalities (eg, +4 or +5, +7, +9, +12, +16) in common, possibly indicating that this cluster of abnormalities might be specific for follicular adenomas. For benign tumors, the three cases showed a remarkably abnormal karyotype. These cases are another example of benign tumors with chromosomal abnormalities that might be specific for follicular adenomas.

Karyotyping and DNA flow cytometry of metastatic ovarian yolk sac tumor.

We karyotyped a metastasis composed of pure yolk sac tumor derived from a primary ovarian germ cell tumor with two components: a dermoid cyst [DNA index (DI) 1.0] and a pure yolk sac tumor (DI 1.88). The metastatic yolk sac tumor had a hypertriploid karyotype and a DI of 1.78 and lacked the germ cell tumor marker i(12p). The absence of this marker in a metastasis from a tumor with a dermoid cyst component might be indicative for a pathogenesis of the yolk sac tumor similar to that of a dermoid cyst and different from that of dysgerminoma.

Cytogenetic study of a nodular hyperplasia of the thyroid after irradiation for Hodgkin's disease.

We describe cytogenetics of a case of nodular hyperplasia of the thyroid with papillary microcarcinoma following radiotherapy for Hodgkin's disease. The chromosomal pattern found was very heterogeneous with a clonal abnormality of chromosome 10, among others. Together with some recent data from the literature, this finding may point to an important role of chromosome 10 abnormalities in the pathogenesis of benign and malignant thyroid neoplasms.

Cytogenetic analysis of the mature teratoma and the choriocarcinoma component of a testicular mixed nonseminomatous germ cell tumor.

We karyotyped two histologically distinct components with different metastatic behavior of a testicular nonseminomatous germ cell tumor. The two components showed an almost identical chromosomal pattern. These almost identical karyotypes of the two components with different metastatic potential suggest that the difference in biologic behavior might result from subtle differences (on microscopic or submicroscopic level) in chromosomal pattern or that these differences are predominantly epigenetically determined and depend primarily on the lineage of differentiation of the tumor component. Trophoblastic differentiation results in an aggressive, angioinvasive tumor but in development of teratoma in a tumor with low malignant potential.

Cytogenetic analysis of the mature and immature teratoma components of a metastatic testicular nonseminomatous germ cell tumor.

A case is described in which the mature and immature teratoma components of metastases of the same testicular nonseminomatous germ cell tumor were karyotyped. The highly similar karyotypes of both components suggest that the phenotypic difference is predominantly epigenetically determined.

Cytogenetic abnormalities and clinical stage in testicular nonseminomatous germ cell tumors.

To study the impact of chromosomal abnormalities on the clinical behavior of testicular nonseminomatous germ cell tumors (TNSGCTs), we compared the chromosomal constitution of primary tumors of patients who initially presented and remained without metastases to those with metastatic disease. Furthermore, the chromosomal pattern of primary TNSGCTs was compared to ploidy and the clinicopathologic risk factors histology and small-vessel invasion. The modal chromosome number and the ploidy were in agreement. No correlation was found between the modal chromosome number and histology, presence of vascular invasion, or clinical stage. No correlation was found between structural chromosome abnormalities, like the number of copies of the i(12p) chromosome, and clinical stage. No obvious differences were found in chromosomal constitution of metastatic and non-metastatic tumors. The results of the present study suggest that in TNSGCTs differences in clinical behavior are not associated with gross chromosomal differences.

Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii).

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42-48 h in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 microg mL(-1) porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4-8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18-30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1-100 microg mL(-1), prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 microg mL(-1)) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.

Effects of repeated superovulation and surgical oocyte collection on ovarian response and natural breeding ability of the tammar wallaby (Macropus eugenii).

The aim of this study was to assess the response of a marsupial, the tammar wallaby (Macropus eugenii) to repeated superovulation and surgical oocyte collection and monitor any effects on subsequent natural breeding ability. Animals (n = 5 per group) were superovulated once, twice or three times with pig FSH (pFSH; 6 mg administered twice per day for 4 days) followed by 4 mg pig LH (pLH). There was an interval of either 5-6 weeks (n = 9) or 12 weeks (n = 1) between the first and second superovulation and 13-17 weeks (n = 5) between the second and third superovulation. Oocytes were collected surgically after each treatment. Serum was collected at the time of each treatment to monitor the formation of anti-pFSH and anti-pLH antibodies. Animals were allowed to mate naturally in the season following superovulation treatment(s). There was no significant difference between groups in the number of large follicles (2-5 mm diameter, mean +/- standard error) produced in response to the first (21.2 +/- 4.3), second (18.0 +/- 6.5) or third (29.0 +/- 4.9) superovulation treatment. Eggs were recovered from approximately 80% of follicles that were flushed during laparotomy. There were significant concentrations of anti-pFSH and anti-pLH antibodies (P < 0.05) detected in previously superovulated animals at the time of the second superovulation but not at the time of the third superovulation. The anti-gonadotrophin antibodies present at the time of repeated superovulation did not cause a significant decrease in average number of follicles. All animals produced pouch young in the breeding seasons after repeated superovulation. Combined with other reproductive technologies, repeated superovulation has the potential to increase the production of offspring from rare or valuable marsupials in captivity.

Chromosomal analysis and the classification of soft tissue sarcomas.

Seventeen soft tissue sarcomas were karyotyped and the chromosomal findings were compared with those in the literature. Some cases were easily classified (immuno)histologically, whereas others defied classification. In four cases with unequivocal histology, a "characteristic" chromosomal abnormality was found. In one case a diagnosis that was doubtful on histologic grounds was supported by the chromosomal findings, whereas in three other cases, the chromosomal pattern led to reconsideration of the histologic diagnosis. The remaining cases either showed chromosomal abnormalities with hitherto undescribed breakpoints or breakpoints described in tumors with different histology. The results extend the knowledge of chromosomal abnormalities in soft tissue sarcomas. Most importantly, the present approach demonstrates that close collaboration between the surgical pathologist and the cytogeneticist may improve the classification of soft tissue sarcomas.