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)

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.

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.

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.

Growth of an implanted fibrosarcoma in rats is associated with high levels of plasma prostaglandin-E2 and thromboxane-B2.

Growth of BN175, a malignant fibrosarcoma, was correlated with high plasma TXB2 and PGE2 levels. This statistically significant increase was first detected 17 days after inoculation of the tumor, at which time the tumors were 20 mms in diameter. A further increase in tumor size was associated with still higher PGE2 and TXB2 values. At the same time, progressive alterations in platelet function, as measured by ADP-induced platelet aggregation, were observed. 6-keto-PGF1 alpha levels remained normal throughout the whole experiment. It was concluded that tumor growth was associated with changes in PG synthesis and platelet function, although it remains unclear whether these changes were caused by some host immunological response towards the tumor or were predominantly the result of tumor PG-synthesis.

Joint report of the Third International Workshop of Canine Immunogenetics. II. Analysis of the serological typing of cells.

Using a standardized microlymphocytotoxicity assay, seven international laboratories evaluated 144 anti-dog lymphocyte antigen (DLA) sera in 319 mixed breed and 152 Beagle dogs. The workshop confirmed the serological definitions for DLA-A2, A3, A9; DLA-B4, B5, B6, B13; DLA-C11(Cwl); and C12(Cw2). Two new specificities were assigned to the DLA-A locus (Aw14 and Aw15) in only the mixed breed dogs. A third specificity (Cw3), was assigned to the DLA-C locus. The antigen and gene frequencies of these alleles differed between the two groups of dogs, but the frequencies of the "blank" were similar in both groups. Future international collaborations will be necessary to definite more completely the polymorphisms of the major histocompatibility complex (MHC) of the dog. Those efforts will benefit from the standard serological test established in this workshop.

Joint report of the Third International Workshop on Canine Immunogenetics. I. Analysis of homozygous typing cells.

The Third International Workshop on Canine Immunogenetics involved 80 potentially DLA-D homozygous typing cells obtained from dogs of various breeds and submitted from five laboratories in Europe and the United States. Mutual reactivity of all cells was studied in mixed leukocyte cultures, and stabilized relative responses were used for analysis. Intralaboratory and interlaboratory comparisons of results suggest that a stabilized relative response of 30% represents an acceptable parameter for "typing responses" indicating phenotypic DLA-D identity of stimulator and responder cells. Using this criterion, 10 clusters of homozygous typing cells were defined and accepted on an international level, and they were assigned the specificities Dw1 to Dw10. At least six additional (provisional) specificities were recognized that were well characterized within individual laboratories but require additional testing before workshop specificities can be assigned. These data show that DLA-D typing is feasible and represents a useful tool in the genetic analysis of the canine major histocompatibility complex. Much work is needed to confirm the present results in family studies, to determine gene frequencies, and to analyze at a molecular level the antigens responsible for mixed leukocyte culture reactivity.

Clinical and immunological evaluation of 20 patients with advanced colorectal cancer treated with high dose recombinant leukocyte interferon-alpha A (rIFN alpha A).

A total of 20 patients with advanced colorectal cancer received recombinant leukocyte interferon-alpha A (rIFN alpha A) either chronically (group I: twice a week up to 20 X 10(6) IU/m2 i.m.) or cyclically (group II: 1-4 periods of 8 consecutive days up to 20 X 10(6) IU/m2 i.m. daily at 20-days intervals) over a period of 12 weeks. There was 1 partial response, 1 mixed response and 1 patient with stable disease, whilst 17 patients had progressive disease. Median survival was 15.5 months. Survival was significantly shorter when the extent of hepatic disease was greater than 25% (P = 0.05), extrahepatic disease was extensive (P less than 0.005), alkaline phosphatase level was greater than 2 X normal (P less than 0.02), or performance status was less than 100% (P less than 0.001). Toxicity consisting mainly of fever, fatigue, anorexia and weight loss was serious in group I and minimal in group II. Administration of rIFN alpha A led to a "short lived" augmentation of natural killer (NK) cell activity. In the cyclically treated group this was a recurrent phenomenon whereas a marked lasting depression of NK cell activity was seen in chronically treated patients. Interferon-gamma production capacity was significantly stimulated during rIFN alpha A therapy. The differences in toxicity and immunostimulatory effects between the two schedules may be of importance in the design of further studies.

Effects of treatment with azathioprine and cyclosporin A on interferon-gamma production by peripheral blood leukocytes of renal allograft recipients.

We have studied the concanavalin A (ConA)-induced interferon gamma (IFN-gamma) production of peripheral blood mononuclear cells (PBMC) of renal allograft recipients. Both under immunosuppressive treatment with azathioprine and with cyclosporin A (CsA) the PBMC of these patients proved deficient for IFN-gamma production when compared to those of healthy controls. After conversion from conventional azathioprine to CsA medication the ConA-induced IFN-gamma production increased.

Leukocyte adherence inhibition, natural killer cell activity, gamma-interferon production capacity and phytohemagglutinin response in patients treated with recombinant leukocyte A interferon.

10 patients with disseminated colorectal cancer were treated either chronically or cyclically with human recombinant leukocyte A interferon (IFl-rA) for 3 months. During this period, leukocyte adherence inhibition (LAI), natural killer (NK) cell activity, concanavalin A-induced gamma-interferon production capacity (GIPCA) and phytohemagglutinin response were sequentially monitored. In both chronically and cyclically treated patients, IFl-rA therapy led to a 'short-lived' augmentation of NK cell activity. In the chronically treated patients, there was a further depression in the NK cell activity during the course of therapy. The outcome of LAI remained unaltered irrespective of the mode of IFl-rA therapy. There was an inverse correlation between GIPCA and phytohemagglutinin response.

Mitogenic responses of canine peripheral blood lymphocytes to staphylococcal protein A.

In order to produce long term lymphoid cell cultures from canine lymphocytes of known histocompatibility antigen specificities, mitogenic responses to staphylococcal protein A (SpA) were examined and compared with those of phytohaemagglutinin (PHA) and concanavalin A (Con A). SpA was found to be the strongest mitogen tested with significant responses to concentrations as low as 31 ng/ml. There was a decrease in responsiveness above optimal mitogen concentrations with SpA and PHA. Peak responses were observed at lower concentrations for longer incubation times. PHA showed a rapid fall off in thymidine uptake below optimal concentrations whereas the SpA dose-response curve was less steep and a shoulder or secondary peak of activity was observed at low SpA concentrations in some cases. Continuous SpA stimulation of lymphocyte cultures resulted in an initial period of cell proliferation followed usually by a second period of cell proliferation around week 7 of culture. To date, viable cell cultures have been maintained for up to 12 weeks in vitro. SpA lymphoblast cultures behave normally in microcytotoxicity tests for serologically defined DLA histocompatibility antigens and remain functional in natural killer (NK) and PHA induced cell mediated cytotoxic reactions against 51Cr-labelled tumour target cells but were not themselves susceptible as target cells for NK activity.