A new high-quality set of singly ((2) H) and doubly ((2) H and (18) O) stable isotope labeled reference waters for biomedical and other isotope-labeled research.

RATIONALE: Research using water with enriched levels of the rare stable isotopes of hydrogen and/or oxygen requires well-characterized enriched reference waters. The International Atomic Energy Agency (IAEA) did have such reference waters available, but these are now exhausted. New reference waters thus had to be produced in sufficient quantity, and higher characterization quality was desired. METHODS: The reference waters have been prepared gravimetrically from three parent waters: natural water, pure (2) H water and highly (18) O-enriched water. These parent waters have been thoroughly assessed for their full isotopic compositions. To ensure the integrity and correctness of the gravimetric procedure, validation measurements have been carried out on the isotopic composition of the produced reference waters by two of our laboratories. These measurements corroborate the values obtained on the basis of gravimetric data. RESULTS: Two new sets of three reference waters enriched in the stable isotopes have been produced and certified: one set of singly labeled waters, only enriched in (2) H, and another set of Doubly Labeled Waters, enriched in both (2) H and (18) O. They cover δ(2) H and δ(18) O values in the range of 800-16000 ‰ and 100-2000 ‰, respectively. The process has led to highly accurate isotopic values for these waters. CONCLUSIONS: These reference waters are now available (called IAEA-604 to IAEA-609). They will be valuable as reference materials for all fields using isotope labeling of water, most prominently, but not exclusively, biomedical research (body composition analyses, metabolic rate measurements). The two waters with the lowest enrichments will also be useful as anchor values for isotope measurements around the natural range.

Doubly Labelled Water analysis: preparation, memory correction, calibration and quality assurance for δ2H and δ18O measurements over four orders of magnitudes.

RATIONALE: The Doubly Labelled Water (DLW) method is an established way of determining the metabolic rate in humans and animals, with the advantage that the subjects need not be confined. The method, however, needs accurate determination of both the δ(2)H and the δ(18)O isotope values over a wide range of enrichments. METHODS: In this paper we describe a number of crucial steps in the process of isotope determination in body fluids. These steps include micro-distillation, correction of the measurements for sample-to-sample memory and calibration of the isotope scales over many orders of magnitudes. In contrast to several published protocols and guidelines, we also take highly enriched samples into account, as they are required for studying the metabolic rate of birds and small mammals. For our isotope scale calibration, we made a set of gravimetrically prepared, double labelled waters with known isotope values. Our quality assurance includes a scheme for easy calculation of the error propagation, leading to a reliable estimate of the analytical error in the metabolic rate. RESULTS: Our memory correction algorithm assumes the existence of three water "pools" that have different sizes and exchange rates with the injected samples. We show that the method can correct even huge memory signals, without the need for "true" values. CONCLUSIONS: With the presented building blocks, we show how to assure a reliable and accurate isotope analysis for the DLW method, both for human and for animal applications. Although our measurements have been performed using isotope ratio mass spectrometry, most of the procedures are also useful for laser spectrometry.

Complementation of the genetic defect in Gunn rat hepatocytes in vitro by highly efficient gene transfer with cationic liposomes.

In this article, we report complementation of the genetic defect of isolated Gunn rat hepatocytes by a highly efficient method for lipofection. Transfections were performed 24 h after plating by using the cationic liposome DOTAP. On average, transfection efficiencies of 21% lacZ+ cells with Wag/Rij rat cells and 27% lacZ+ cells with Gunn rat cells could be obtained when the parenchymal cells were transfected in a hormone-defined, serum-free medium. LacZ expression vectors with the CMV promoter were more effective than constructs containing the RSV or the TK promoter. A linear relationship between the viability of hepatocytes after isolation and the percentage of lacZ+ cells was observed with both rat strains, with a maximum of 40% lacZ+ cells at a viability of 94%. The transfection efficiencies were significantly lower in the absence of growth factors, in dexamethasone-containing medium, or when serum was present during plating. Our data are consistent with the assumption that a mitotic event is required for efficient lipofection. Bilirubin conjugation activity could be detected in microsomes from Gunn rat hepatocytes after transfection with two different B-UDPGT expression constructs. Highest conjugation activity was achieved with a vector containing a terminal intron. With this construct on average 4% of the bilirubin conjugation activity of normal human liver microsomes could be achieved in total microsomes of transfected Gunn rat hepatocytes. The implications of our data for gene therapy of hepatic disease with nonviral vectors, in particular bilirubin conjugation deficiency (Crigler-Najjar disease) are discussed.

Correction of an inborn error of metabolism by intraportal hepatocyte transplantation in a dog model.

The aims of this study were (1) to assess portal hemodynamics during intraportal hepatocyte transplantation (HTX) in dogs, (2) to evaluate a new method for the detection of transplanted hepatocytes using 5-bromo-2'-deoxyuridine (BrdU) incorporation, and (3) to determine the metabolic effects of HTX on an inborn error of the purine metabolism in dalmatian dogs. HTX was performed by intraportal infusion of freshly isolated allogeneic beagle hepatocytes. Portal flow and pressure were monitored continuously during HTX. For the detection experiments, beagles received hepatocytes that had been exposed to BrdU during regeneration of the donor liver, induced by partial hepatectomy. For metabolic studies, dalmatian dogs were used as recipients. Repetitive HTX was performed. As judged by the portal hemodynamics, the number of hepatocytes that could be infused safely varied from 5 x 1O(8) to 8 x 1O(8) in beagles, to 1 x 10(9) in dalmatians. Transaminase levels showed a 5- to 6-fold increase (P=0.05) after HTX, but normalized within 3 weeks. BrdU-positive cells were identified in the recipient livers 2 weeks after HTX and 5-10% of the total amount of transplanted hepatocytes was retrieved. A significant (P=0.05) decrease in serum uric acid was demonstrated after repeated HTX in dalmatians. In conclusion, (1) intraportal HTX is feasible, but portal hypertension limits the maximum amount of hepatocytes that can be infused in one HTX; (2) BrdU labeling is an attractive method for the detection of transplanted hepatocytes in the recipient liver; and (3) after two consecutive hepatocyte transplantations, a temporary correction of the purine metabolism was accomplished in the dalmatian dog.

The effect of partial hepatectomy on tumor growth in rats: in vivo and in vitro studies.

Residual tumor in the remnant liver after partial hepatectomy (PH) for colorectal liver metastases is a serious clinical problem. This fact is reflected by the high number of recurrences after potentially curative liver resections. Liver regeneration, it appears, might influence the growth of remaining micrometastases in the liver. Using rats, we demonstrated enhancement of growth of a syngeneic colon carcinoma (CC 531) in the remnant liver after 70% PH. Fourteen days after PH, tumor weights in the liver were twice as high as those of sham-operated rats. This difference in tumor weight was not found in extrahepatic tumors. In vitro experiments did not show stimulation of cultured CC 531 cells by portal or systemic serum withdrawn 24 hours or 14 days after hepatectomy as compared with sera obtained after sham operation. Co-cultures of CC 531 cells and hepatocytes (in ratios of 1:10 or 1:1) demonstrated a higher 3H-thymidine incorporation than was the case in separately cultured cells. In co-cultures, bromodeoxyuridine (BrdU) incorporation in DNA was found primarily in CC 531 cells and rarely in hepatocytes. Cell density appeared to be of influence on 3H-thymidine incorporation in co-cultures. Hepatocytes were found to have a stimulating effect on CC 531 cells in low-density cultures, whereas high-density cultures exhibited an inhibiting effect after a culture time of 120 hours. These results show that, depending on cell density in co-cultures, a paracrine stimulating influence of hepatocytes on this type of colon carcinoma cells (CC 531) might be responsible for the increased tumor growth in vivo.

Effects of high-energy shockwaves on normal human fibroblasts in suspension.

To gain insight in the effects of shockwaves on human cells the relationship between the energy density and the number of shockwaves as well as their effect on suspensions of normal cells was studied. At energy densities of 0.37, 0.6, 0.78, and 1.20 mJ/mm2 fibroblasts were subjected to 50, 100, 250, 500, and 1,000 shockwaves. Each test was performed three times and one sample was used as control. A decrease in viability related to the logarithm of both the number (P = 0.0000) and the energy density (P = 0.001) of the shockwaves was statistically demonstrable 1 hr after the shockwave application. The energy density of the shockwaves has less influence on the viability than the number of applied shockwaves. Seeding of viable cells 1 hr after the shockwave application showed that the decrease in the 48-hr growth potential was statistically dependent of the number of applied shockwaves only (P = 0.0007). After 24 hr no difference in the 48-hr growth potential could be demonstrated between viable shockwave-treated cells and control cells. The literature as well as our own investigations in vitro and in vivo indicate that shockwaves have a logarithmic dose-dependent destructive effect on cells in suspension, but they also seem to have a dose-dependent stimulating influence on the healing process in damaged tissues. Due to the logarithmic relationship between the viability and both the number and energy density of the applied shockwaves it might be expected that even excessive numbers of high-energy-density shockwaves don't soon lead to total destruction of all cells in the suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative response of hepatocytes transplanted into spleen or solid support.

Understanding the regenerative behavior of transplanted hepatocytes is of great importance for developing and improving such novel therapeutic strategies as hepatocellular transplantation and ex vivo gene therapy. In this study the proliferative responsiveness of transplanted syngeneic rat hepatocytes was examined in relation to the timing of the administration of a mitogenic stimulus. For this purpose nuclear bromodeoxyuridine incorporation after partial hepatectomy was investigated during the early post-transplant phase. The response of intrasplenically transplanted hepatocytes was compared to that of liver cells engrafted in polytetrafluoroethylene solid supports that had been implanted intraperitoneally 4 weeks prior to transplantation. Nonstimulated, engrafted hepatocytes exhibited a labeling index of approximately 0-1% independent of the transplantation technique used. This "spontaneous" labeling index did not change with time. Partial hepatectomy, executed simultaneously with hepatocyte transplantation through either technique, did not result in significant alteration of this proliferation index. Delayed kinetics were found not to be responsible for this lack of responsiveness. When the mitogenic stimulus was given between 2 and 6 weeks post-transplantation, a significant increase in labeling index was observed in comparison to sham-treated control animals. Maximal labeling indices of approximately 3-4% were found if the stimulus took place at 4 weeks post-transplantation. Both the pattern and the extent of the proliferative response seen in liver cells engrafted in solid supports were similar to the ones found in intrasplenic hepatocytes, indicating adequate vascularization of the supports. This data provides the first description of proliferative response in hepatocytes transplanted by the solid support technique, which may offer an attractive alternative to the intrasplenic route.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells.

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h 51Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h 51Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.

Evidence of metabolic activity of adult and fetal rat hepatocytes transplanted into solid supports.

This study was undertaken to assess the metabolic effect of fetal and adult hepatocyte transplantation in the Gunn rat, genetically incapable of bilirubin conjugation. A comparison was made between fetal and adult hepatocytes transplanted into the spleen, and those injected into polytetrafluoroethylene (PTFE) solid supports that had previously been implanted intraperitoneally. Between 4 and 12 weeks after intrasplenic transplantation of adult liver cells, serum bilirubin was significantly decreased when compared with control animals (39.6 +/- 5.6%; P less than 0.01 vs. controls). Intrasplenic transplantation of fetal hepatocytes resulted in a maximal decrease of 33.2 +/- 9.1% at 8 weeks postoperatively (P less than 0.02 vs. controls). Similar declines of serum bilirubin levels were found after transplantation of adult or fetal liver cells into the solid supports. At 12 weeks after transplantation, bilirubin conjugates were detectable in the bile of all animals that underwent intrasplenic hepatocyte transplantation and in 60% of those that underwent the solid support procedure, whereas none could be detected in control animals. Histological evidence of surviving cells was obtained in all but one animal at 12 weeks, and confirmed at 12 months postoperatively. It is concluded that the PTFE solid support technique offers an attractive alternative to the intrasplenic route, and that both fetal and adult hepatocytes, transplanted in either way still exert their conjugating activity after 12 weeks.

Eicosanoids in breast cancer patients before and after mastectomy.

In 19 patients with a malignant breast tumor, tumor tissue and blood were taken to determine the eicosanoid profile and platelet aggregation. Values were compared with those of patients with benign tumors (n = 4), or undergoing a mammary reduction (n = 7). Postoperatively, blood was taken as well in order to compare pre- and postoperative values. Eicosanoids were measured in peripheral blood monocytes and mammary tissue by means of HPLC; furthermore, TXA2, 6-keto-PGF1 alpha, and PGE2 were determined by RIA. Differences in pre- and postoperative values of cancer patients were seen in plasma RIA values: PGE2 and 6-k-PGF1 alpha were significantly higher preoperatively when compared with postoperatively, however, such differences were seen in the control groups as well. Compared to benign tumor or mammary reduction test material the eicosanoid profile of tissue obtained from malignant mammary tumors showed important differences. Except for PGF2 alpha, HHT and 15-HETE no detectable quantities of eicosanoids were found in the non-tumor material, whereas in the malignant tumor material substantial quantities of a number of eicosanoid metabolites were present. Statistically significant correlations could be established between patient/histopathology data and the results of the platelet aggregation assays, e.g. between menopausal status and ADP aggregation; oestrogen receptor (+/-) and collagen and arachidonic acid aggregation, inflammatory cell infiltration score and arachidonic acid aggregation and fibrosis score and ADP aggregation. The results show that eicosanoid synthesis in material from mammary cancer patients is different from that in benign mammary tissue. The implications, in particular, in relation to future prognosis of the patient, remain obscure.

Comparative cell culture effects of shape memory metal (Nitinol), nickel and titanium: a biocompatibility estimation.

Nitinol is an equiatomic alloy of nickel and titanium which has been attracting increasing interest in the field of biomedical engineering. To quantify toxicity as a preliminary evaluation of biocompatibility, inhibition of mitosis in human fibroblasts in tissue cultures exposed to test materials is an accepted screening method, although a dose-effect relationship had never been investigated. In this experiment, the effect of an increasing dose exposure to Nitinol, nickel or titanium on human fibroblasts in cell cultures was tested in subgroups in comparison with a control group. The results showed that nickel induces a significant (p < or = 0.05) inhibition of mitosis in human fibroblasts, whereas no significant effects of this kind were found for titanium or Nitinol. According to the results of these studies, Nitinol is to be considered in this respect biocompatible and comparable to titanium, which would seem to justify application as a surgical implant.

Eicosanoid profile of healing colon anastomosis and peritoneal macrophages in the rat.

Because intraperitoneal administration of prostaglandin E2 (PGE2) has a negative influence on the healing of colonic anastomosis, the production of eicosanoid products in the healing rat colon after resection and anastomosis was studied using high performance liquid chromatography. Normal colonic tissue metabolizes small amounts of arachidonic acid into cyclo-oxygenase and lipoxygenase products. After construction of an anastomosis, however, there is increased production of lipoxygenase products, while cyclooxygenase activity remains low. Increased amounts of PGE2 and other cyclo-oxygenase products are not produced after anastomosis of the colon and probably do not play a major role in uncomplicated healing of the large intestine in the rat. During the first eight days of repair in the anastomosed colonic tissue, a statistically significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was found compared with control colon tissue (p = 0.001). At the same time peritoneal macrophages from these rats showed increased 12-HETE production. Eicosanoid synthesis of peritoneal macrophages resembled eicosanoid synthesis of anastomosed colon taken from the same rat indicating that 12-HETE, in particular, may be of macrophage origin.

Immunomodulation by recombinant rat interferon-gamma in vivo.

Most studies on the immunomodulating effects of interferon-gamma (IFN-gamma) have been performed in vitro, using recombinant mouse and human IFN-gamma preparations. Recently, recombinant rat IFN-gamma (rRIFN-gamma) became available, enabling extensive studies with this new preparation in vivo. In the present study a LEW rat model was used to determine the efficacy of rRIFN-gamma on immune functions in vivo. LEW rats were treated with rRIFN-gamma by continuous intravenous infusion at a dosage of 1.5 x 10(5) U/kg.h for 2 consecutive days. Twelve hours after cessation of rRIFN-gamma administration immune functions, including NK-cell activity, phagocytosis, and mitogen-induced blastogenesis, were assessed. All experimental animals displayed a marked reduction in the number of peripheral blood and bone marrow cells when compared with controls (p less than 0.005). Assessment of immune functions revealed a significant enhancement of NK-cell activity (p less than 0.001), phagocytosis (p less than 0.05), and mitogen-induced blastogenesis (p less than 0.05). These findings indicate that rRIFN-gamma, when given in high dosages, has a stimulatory effect on various immune functions, which substantiates its important immunological role in vivo.

Synthesis of 18F-labelled VP 16-213 and podophyllotoxin using acetyl hypofluorite.

The fluorination of the antitumour agents VP 16-213 and podophyllotoxin using [18F]acetyl hypofluorite is described. Overall labeling yields of 10% and 20% respectively were obtained. The F-atom is found to be introduced into the E-moiety of the compounds, apparently without unwanted conformational changes.

Changes in immunological parameters after conversion from cyclosporine A to azathioprine in renal transplant recipients.

Long term CsA therapy did not interfere with the basal levels of natural killer (NK) activity in stable cadaveric renal transplant recipients. However, 3 months after changing immunosuppressive therapy from CsA to AZA, NK activity was significantly decreased (36 +/- 25% vs 19 +/- 15%, P less than 0.01). Following in vitro exposure to IFN-gamma an increase in NK activity from 36 to 44% (P less than 0.05) could be induced during CsA therapy but this was no longer observed after conversion to AZA (19 to 22%, N.S.). A prominent decline in the number of NK cells expressing the surface receptor for the Fc portion of IgG was also found postconversion. The IFN-gamma production capacity after mitogen stimulation of unprimed lymphocytes was more depressed during CsA than during AZA therapy (median 25 vs 80 U/ml 10(6) cells, P less than 0.05), suggesting a reversible inhibition of CsA on lymphokine production. Despite the better IFN-gamma production capacity, both the activity, inducibility and number of NK cells were significantly lower under AZA therapy than under CsA therapy. These findings indicate that CsA exerts its immunosuppressive action without an important interference with NK activity. Monitoring mononuclear cells showed a decrease in absolute numbers of all phenotypically distinct cells studied after conversion. The prominent decrease in CD 8 cells resulted in an increase of CD 4/CD 8 ratio.

Inhibition of tumor metastasis by carrageenan-induced granulomas.

At -1, 0, +1 weeks from tumor inoculation, carrageenan-impregnated cotton sponges were subcutaneously implanted. Tumor BN472, a malignant adenocarcinoma, was transplanted in syngeneic Brown Norway female rats, either subcutaneously or intravenously. Plasma eicosanoid values (prostaglandin-E2, thromboxane-B2 and 6-keto-prostaglandin-F1 alpha) were determined as well as the cellular immune response (natural killer activity, concanavalin-A and phytohemagglutinin stimulation of spleen lymphocytes). Primary tumor growth and the number of tumor foci in the lungs were measured as parameters of tumor growth and dissemination. No statistically significant differences were observed in primary tumor growth. However, the number of metastatic foci in the lungs of rats in which the tumor was implanted subcutaneously, as well as those in which the tumor was inoculated intravenously, was significantly reduced in those that had carrageenan implanted one week after tumor inoculation. In all other carrageenan-treated groups only slightly reduced numbers of metastatic foci were seen. In those rats with a decreased number of tumor metastatic foci in the lungs, no correlation could be shown with either altered plasma prostaglandin levels, or cellular immune response.

Role of alveolar macrophages in tumor-bearing rats: tumoricidal properties of carrageenan-activated macrophages.

Tumor BN472, a malignant mammary adenocarcinoma, was subcutaneously transplanted into syngeneic female Brown Norway rats. Seven days after tumor inoculation, carrageenan-impregnated synthetic sponges were subcutaneously implanted in control and tumor-bearing rats. Another week later the animals were sacrificed and alveolar macrophages were harvested and tested for tumoricidal activity against a tissue culture line of BN472 cells and their capacity to phagocytose formaldehyde-treated sheep erythrocytes. The data demonstrate that carrageenan statistically significantly enhances the tumoricidal activity of alveolar macrophages in tumor-bearing rats. Phagocytic activity of the macrophages in these animals is not different from sham-operated control animals, whereas the phagocytic activity of tumor-bearing rats is statistically significantly decreased.

Omega-3 fatty acids inhibiting the growth of a transplantable rat mammary adenocarcinoma.

BN/Bi inbred female rats fed diets containing different amounts of polyunsaturated fatty acids, either of the omega-3 or omega-6 type, each received an implant of a syngeneic mammary adenocarcinoma. When the diameter of the tumors reached 20 mm, they were surgically removed; 2 weeks thereafter the animals were sacrificed and lung metastases were counted. Cellular immune response was determined before tumor inoculation; certain prostaglandin values in plasma and platelet aggregation were measured before and after tumor inoculation. Plasma prostaglandin E2 and thromboxane B2 values were significantly decreased in those rats fed a diet containing menhaden oil. 6-Keto-prostaglandin F1 alpha, cellular immune response, and platelet function were not significantly different in either one of the diet groups. Tumor growth in the groups of rats receiving the omega-3 fatty acids in their diet was significantly inhibited in comparison with that in the rats receiving the omega-6 fatty acids. However, the number of metastases was not significantly altered.