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In 30 consecutive children undergoing cardiac surgery, two different types of fluid were given randomly for volume replacement in the pre-bypass period. In group 1 (n = 15), low molecular weight hydroxyethyl starch solution (LMW-HES) (6% HES; mean molecular weight 200,000 Da, molar substitution 0.5) and in group 2 (n = 15) 20% albumin (HA) was infused from the induction of anaesthesia until the start of cardiopulmonary bypass (CPB). In addition to haemodynamic values, various laboratory variables were measured before and after CPB until the morning of the 1st day after operation. The patients did not differ in diagnosis and conduct of CPB (lowest rectal temperatures: group 1 29.0 (SD 1.1) degrees C; group 2 29.4 (1.0) degrees C). Haemodynamic data (MAP, HR, CVP), anti-thrombin-III, fibrinogen, platelet count and coagulation variables were comparable between the groups until the 1st day after operation. Postoperative blood loss and the use of homologous blood or blood products were similar in all children. Albumin concentration increased after infusion of albumin (35-47 g litre-1) and was significantly greater until the end of the operation compared with the LMW-HES-treated children. Colloid osmotic pressure, however, was similar in the two groups and returned to baseline values on the 1st day after operation (LMW-HES group 19.31 (1.2) mm Hg; HA group 18.0 (1.3) mm Hg). Post-bypass urine output and creatinine values also did not differ between the groups. Anaphylactic reactions were not observed in any of the patients. It can be concluded that LMW-HES solution can be used effectively and safely for volume replacement in the pre-bypass period in small children undergoing cardiac surgery.

We determined loss of heterozygosity from formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using microsatellite polymorphism. The polymorphism was assayed based on DNA amplification by the polymerase chain reaction (PCR). The PCR-analyzed microsatellite method was applied to assay degraded DNA extracted from paraffin-embedded blocks with adenocarcinoma of colon. The DNA from 26 tumors as well as their corresponding normal tissue samples were successfully amplified using a dinucleotide microsatellite located within an intron of the deleted in colorectal carcinoma gene. Allele losses on this marker were detected in 33% of informative colorectal carcinomas. This study demonstrates that microsatellites provide a powerful set of DNA markers for loss of heterozygosity on archival specimens.

Colorectal carcinogenesis is a complex multistage process and occurs through the accumulation of gene mutations in both oncogenes and tumour suppressor genes. Frequent genetic abnormalities include mutation of the familial adenomatous polyposis (APC) and/or the mutated in colorectal cancer (MCC) genes on chromosome 5q21, activation of K-ras and loss of the tumour suppressor genes p53 and DCC (deleted in colorectal cancer). In our laboratory we have developed human in vitro colonic cell culture model systems, to determine the biological consequences of these well characterised genetic changes, and how such changes can uncouple proliferation from differentiation and ultimately lead to the malignant phenotype.

The task of purifying monoclonal antibodies (MAbs) and human recombinant antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active recombinant antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg/cm2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 micrograms/cm2 for the amine-modified and 39 micrograms/cm2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A-Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse ATIII/IIa and HCII/IIa interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.

In order to attempt to examine the question of whether papillary cystic tumors (PCT) of the pancreas represent a hormone-dependent neoplasm or not, three cases of PCT were biochemically and immunohistochemically investigated. The patients included a 27-year-old man, a 14-year-old girl and a 21-year-old female. Two of the three patients showed measurable biochemical levels of progesterone receptor in the tumors by the dextran-coated charcoal (DCC) method, and an immunohistochemical study revealed that the nuclei of the tumor cells in all the three cases were diffusely positive for progesterone receptor. In the literature, there have been more than 300 female patients of PCT with a significant minority of only twenty male patients, and the mean age of the cases with PCT in men (31.4 years old) is higher than that in women (25.5 years old) (P < 0.05). These results therefore suggest that PCT is a sex hormone-dependent neoplasm.

EGF-induction of human astrocytoma and A431 cells leads to c-fos transcriptional activation and then repression. This could be correlated with changes in the DNA binding characteristics of the c-fos regulatory protein ternary complex factor (TCF) present in nuclear extracts from these cells. Band shifts showed the appearance of induction-related slowly migrating protein-DNA complexes, detected as ternary complexes on the c-fos SRE using a truncated SRF molecule and by direct binding to the Drosophila E74 Ets-protein recognition sequence. By several criteria both types of complexes represented TCF. The appearance of the slow ternary and direct complexes correlated with c-fos transcriptional activation, and their disappearance coincided with the ensuing c-fos shut-off. Blocking c-fos transcriptional repression with the phosphatase inhibitor okadaic acid led to their continued presence. They were sensitive to protein phosphatase 2A but not 1 alpha, and similar slow complexes were formed by partially purified p62TCF phosphorylated by a copurifying kinase activity. Thus the phosphorylation state of TCF correlated strongly with c-fos promoter activity. Since ternary complex formation mediated by full-sized SRF was only slightly affected under comparable conditions, we propose a model for c-fos regulation involving modification of constitutively bound TCF.

We have looked for evidence of coagulation activation in six subjects with haemophilia B by performing a single-blind active control cross-over study comparing a recently developed factor IX concentrate with a conventional prothrombin complex concentrate (PCC). Samples were obtained before infusion and at 0.25, 0.5, 1, 2, 4, 6, 12, 24, 36 and 48 h for assay of factor IX, prothrombin time, fibrinopeptide A (FPA), prothrombin fragment F1 + 2, D-dimer, thrombin-antithrombin complexes (TAT) and antithrombin III (ATIII). Following administration of the PCC there was evidence of coagulation activation in five of the six recipients for up to 6 h after the infusion. The factor IX concentrate induced a moderate degree of coagulation activation in one subject. There was no significant difference between the two products in respect of either recovery or half-life. This study provides further evidence that the new high purity preparations of factor IX concentrates produce significantly less coagulation activation than currently available PCCs. It remains to be established whether this will result in a corresponding reduction in thromboembolic complications in clinical use.

Oligosaccharides of heparin with high affinity for antithrombin III (ATIII) have been immobilized onto surface-modified NHLBI Primary Reference low density polyethylene (PE). PE was modified by radiofrequency plasma polymerized (< 150 nm thick) films derived from N-vinyl-2-pyrrolidone (PPNVP) or allyl alcohol (PPAA), and coupled by chemical derivatization to either 3-aminopropyltriethoxysilane or amino-terminated poly(ethylene oxide). High affinity heparin oligosaccharides (HA-heparin, anti-factor Xa activity of 592 +/- 120 IU/mg) prepared by partial deaminative cleavage of commercial crude heparin and fractionated by agarose-ATIII affinity chromatography, were immobilized to surface-modified PE by reductive amination. The anticoagulant activity, as determined by a chromogenic assay for the inhibition of factor Xa, was estimated to be 30-70 mIU/cm2, with binding estimated to be 56-119 ng/cm2. The highest activity was obtained for the HA-heparin immobilized to PE modified by PPNVP with a PEO spacer. Visual confirmation of ATIII binding to immobilized HA-heparin was demonstrated by a gold-labeled double antibody method with imaging by SEM.

<h4>Background and methods</h4>In a prospective study, the 5-year survival of 57 patients with Stage II endometrial cancer was correlated to the estrogen (ER) and progesterone (PR) receptor concentrations using the dextran-coated charcoal (DCC) technique. The cutoff level was 3 fmol/mg of protein for ER and 6 fmol/mg of protein for PR.<h4>Results</h4>An histopathologic examination of the tumor tissue samples before treatment demonstrated that 16% of the cases (9 patients) were well-differentiated (Grade 1), 45% (26 patients) moderately differentiated (Grade 2), and 39% (22 patients) poorly differentiated (Grade 3). Only 3 were ER negative, whereas 19 were PR negative. The mean concentrations for ER and PR were 106 and 162 fmol/mg of protein, respectively. All patients received standard treatment, including preoperative irradiation. The total 5-year survival rate was 82% (47 of 57 patients). Deep myometrial invasion was important in Grade 3, but not in Grades 1 and 2; in these two grades, all five patients with deep infiltration survived. There was a correlation between receptor concentration and grade, with a significant difference between Grades 1 and 2 versus Grade 3. All patients who were ER negative survived, as did all patients who were PR negative in Grades 1 and 2. Of the patients with Grade 3 disease who died, four of six (67%) were PR negative. Of the patients with Grade 3 disease who survived, 10 of 16 (63%) were PR negative.<h4>Conclusions</h4>Patients who survived for 5 years did not have significantly different ER and PR concentrations than those who died. The mean ER and PR concentrations in patients who survived were 99 and 159 fmol/mg of protein, respectively, compared with 108 and 178 fmol/mg of protein, respectively, for those who died.

Structural diversity enables class Ia molecules to present a diverse repertoire of peptides to the T cell receptor. This diversity is thought to be generated by recombinations between class I genes. We have found that two class Ib Q2 alleles exhibit extremely high sequence diversity, even higher than class Ia alleles. Clustered nucleotide differences between Q2b and Q2k suggest that this sequence diversity was generated by microrecombinations between Q2 genes and other class I genes. The relatively high expression of Q2b in the thymus may be significant and perhaps suggests a novel role for a Q2b product in the education and selection of the T cell repertoire.

The p50 and p65 subunits of NF-kappa B represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo- and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-kappa B can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-kappa B regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein I kappa B.

The putative tumor suppressor gene DCC has been shown to be frequently lost or expressed at low levels in colorectal, gastric, pancreatic, and esophageal carcinomas. In the present study, the DCC gene and its mRNA expression in human and rat prostatic carcinoma cells as well as in prostatic carcinoma tissues were examined by reverse transcriptase-polymerase chain reaction and polymerase chain reaction-loss of heterozygosity. The DCC gene was present and expressed in normal prostatic cells. However, its expression was decreased or undetectable in all prostatic carcinoma cells from either humans (4 cell lines) or rats (5 cell lines). In patients, 12 of 14 cases (86%) showed reduced DCC expression and 5 of 11 informative cases (45%) showed loss of heterozygosity at the DCC locus. These results demonstrate that loss of DCC expression and loss of heterozygosity at the DCC locus are a frequent feature of prostatic carcinoma cells.

Conditioned media from 14 short term fibroblast cell lines were mitogenic for human breast cancer cells with different steroid receptor profiles in serum-free culture. Fibroblast-conditioned medium stimulated tritiated thymidine incorporation in short term culture and growth in a longer proliferation study as measured by the MTT colorimetric assay. Conditioned media from benign and malignant epithelial cells were non-stimulatory for breast cancer cells but that derived from endothelial cells showed similar stimulation to fibroblasts. Partial purification of fibroblast-conditioned medium identified a peptide with a molecular weight of approximately 8 kDa that showed no affinity for heparin and was mitogenic for MCF-7 breast cancer cells.

Heat shock protein 90 (hsp90) is associated with many steroid receptors in tissue homogenates. It is widely accepted that hsp90 regulates the binding of the receptor to the corresponding gene regulatory element. However there is no unequivocal evidence that steroid receptor-hsp90 complexes are present in the intact cells. We demonstrate here the absence of progesterone receptor (PR)-hsp90 complexes in intact target cell nuclei, using immunohistochemical and biochemical methods to determine the location and composition of the nonliganded (aporeceptor) and liganded (holoreceptor) PR complexes. In the chicken oviduct cells, both apo- and holoreceptors were nuclear, while hsp90 was exclusively cytoplasmic. When expressed transiently in HeLa cells, hsp90 was detected in the cytoplasm and PR was detected in the nucleus. Their location or staining intensity was not affected when they were coexpressed in the same cells. To confirm that the sensitivity of the immunohistochemical detection of hsp90 and PR did not differ significantly, a chimeric hsp90-PR was transiently expressed in HeLa cells. Both hsp90 and PR antigens of the chimera were detected in nuclei with the same intensity. In homogenates of the same tissue samples that were used for immunohistochemistry, the PR was complexed with hsp90. Hsp90-PR complexes were formed in vitro when immature bursa of Fabricius, known to contain high levels of hsp90, was homogenized in the presence of hsp90-free aporeceptor, while holoreceptor did not associate with hsp90. Our data show that nuclear PR is not complexed with hsp90 in vivo and suggest that the 8S-PR may be an in vitro artifact generated during tissue processing.