Immunological unresponsiveness to hepatic allografts in rats. Immunological reactivities of the recipient to donor antigens.

Wistar (RT1bv1) rats transplanted orthotopically with ACI (RT1a) livers survive indefinitely without any immunosuppression, while heterotopic heart grafts or skin grafts are rejected acutely in this combination. Levels of alkaline phosphatase after liver allografting remain significantly higher than those found in controls receiving syngeneic grafts. We studied changes in immune responsiveness in rats receiving liver grafts. Local graft-versus-host reactivity was present at all times assayed. Delayed type hypersensitivity reactions were already positive 2 weeks after liver transplantation and increased in strength. Liver graft-bearing rats were subsequently grafted with donor or third-party skin. Third-party skin grafts survived significantly longer on liver-grafted rats than on untreated controls when grafted within the first week after grafting. Donor-type skin grafts survived longer than controls when grafted within the first 4 weeks after liver grafting, although the skin grafts were eventually rejected. Donor-type skin grafted more than 8 weeks after liver grafting was rejected acutely. In an adoptive transfer assay, ACI hearts survived significantly longer in Wistar rats given serum from Wistar donors 2-4 weeks after ACI liver grafts than in untreated controls. On the other hand, spleen cells obtained at any period after liver grafting were not capable of prolonging cardiac allograft survival after transfer to syngeneic recipients. Thus cellular responses to ACI antigen are not changed during the life-span of liver-grafted animals. Evidence suggests that a serum "enhancing" factor protects the donor liver from rejection in the initial period after liver transplantation. The long-term acceptance of liver grafts is discussed.

Clinicopathological study of livers from brain-dead patients treated with a combination of vasopressin and epinephrine.

Studies were made on the pathological lesions and biochemical indices of the livers of 22 patients in whom normal hemodynamics was maintained for 0-48 days after brain death by administration of vasopressin and epinephrine. Thirty-one specimens of liver tissues were obtained by percutaneous biopsy or at autopsy. The degrees of central venous congestion, central fibrosis, focal fibrosis, fatty metamorphosis, piecemeal necrosis, periportal fibrosis, and intrahepatic cholangitis in livers on various days after brain death were compared with those on the day of brain death (day 0). Central venous congestion was extensive on days 0-4, significantly less on days 5-14, and then again extensive on days 15-48. Central fibrosis and focal fibrosis showed no remarkable change during the 48-day period. Fatty metamorphosis, piecemeal necrosis, and periportal fibrosis showed no significant changes until day 16, but spread extensively on days 40-48. Intrahepatic cholangitis was scarcely observed on day 0 but began to increase after day 3, and spread extensively after day 5. The level of serum glutamic pyruvic transaminase did not increase in most patients until day 15. The mean value of prothrombin activity also did not decrease until day 15. However, the mean value of serum alkaline phosphatase increased gradually after day 3, and was correlated with cholangitis. The present study showed that during prolonged hemodynamic maintenance of brain-dead patients, pathological lesions did not spread or diminished and that biochemical indices did not become worse, or improved, in the first 2 weeks, except for increases in cholangitis and the serum alkaline phosphatase level.

Prolonged survival of hamster-to-rat liver xenografts using splenectomy and cyclosporine administration.

The immunosuppressive effect of splenectomy, alone or in combination with cyclosporine (CsA), was examined in hamster-to-rat orthotopic liver xenografts. The mean survival time was 7.3 +/- 0.5 days in untreated controls, 7.6 +/- 0.8 days with 40 mg/kg/day CsA, 7.2 +/- 0.4 days with splenectomy alone, and 17.6 +/- 5.6 days with splenectomy combined with 30 mg/kg/day CsA (P less than 0.01). The longest survival time was 27 days in this group. Marked enlargement of the spleen and high lymphocytotoxic antibody titer were characteristic of the unmodified recipients and those treated with CsA alone. Splenectomy by itself decreased the antibody formation without improvement of graft survival. In animals treated with the combined regimen, the lymphocytotoxic antibody titer was significantly suppressed, and the PMN and round cell infiltration were greatly reduced. Therefore, a synergistic effect was postulated between cyclosporine and splenectomy in this liver xenograft system.

A crucial effect of splenectomy on prolonging cardiac xenograft survival in combination with cyclosporine.

In this study we investigated the effect of splenectomy in combination with cyclosporine (CsA) on survival of heterotopic cardiac xenografts from hamster to rat. A 12-fold prolongation of mean cardiac xenograft survival, to 41 days, was accomplished with the combined therapy. In both untreated controls and CsA-treated recipients rejection occurred in 3 days. Splenectomy by itself prolonged xenograft function to 5 days. Evidence of humoral-mediated rejection in this cross-species combination was given for the extensive thrombosis and hemorrhage in the subepicardial area, the appearance of lymphocytotoxic titers just before graft function ceased, and the presence of IgM deposits in subepicardial vessels of the xenograft. CsA by itself could not modify this pattern of rejection. Splenectomy decreased antibody formation significantly and rejection became more cellular in nature. The regimen of splenectomy in association with CsA suppressed antibody titers and produced a CsA dose-dependent prolongation of xenograft survival. Thus, a complementary or synergistic effect is the result of the immunosuppressive regimen of splenectomy and CsA in hamster-to-rat cardiac xenografts. In this study the effect of splenectomy in controlling the humoral response in concordant xenografts and its role in future clinical xenografting is emphasized.

Evaluation of preoperative and intraoperative arterial stimulation and venous sampling for diagnosis and surgical resection of insulinoma.

BACKGROUND: The precise intraoperative localization of insulinoma is essential for successful surgical treatment. In addition to various imaging modalities developed recently, arterial stimulation and venous sampling (ASVS) has also been used for tumor localization. METHODS: Preoperative and intraoperative ASVS procedures were performed in 6 patients with insulinoma. Intraoperative ASVS was performed before and after tumor resection. Immunoreactive insulin (IRI) concentrations and the IRI ratio (IRI concentration at each time interval after calcium injection/baseline IRI concentration) were determined by the conventional or a quick IRI method. RESULTS: The site of the tumor was identified preoperatively in all patients. The peak of the IRI ratio varied widely, but setting the cutoff value at 3.0 clearly differentiated peak IRA ratios in feeding arteries from those of nonfeeding arteries. Intraoperative ASVS showed a similar elevation of IRI levels, but the elevation disappeared after tumor resection in all but 1 patient. In 2 patients, resection of the tumor was confirmed during surgery by measuring IRI levels by the quick IRI method. CONCLUSIONS: A combination of ASVS and conventional imaging modalities is useful for precise localization of insulinoma. Resection of the tumor can be confirmed intraoperatively by comparing IRI levels associated with preoperative and postresective ASVS.

Prolongation of cardiac allograft survival by intraportal injection of donor antigens--differential mechanisms according to the timing of injection.

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) before transplantation as well as at the time of transplantation significantly prolongs cardiac allograft survival in rats (7). Long-term establishment of chimerism induced by intraportal administration of SPCs could be a part of this prolongation. In this study, we examined the effect of irradiation of SPCs as a source of donor antigens on cardiac allograft survival. Experiments were carried out using DA (RT1a) as the donor and BUF (RT1b) as the recipient strain. Fifty million irradiated or nonirradiated SPCs were injected either intravenously (i.v.) or intraportally (i.p.) on day -10 or day 0, the day of cardiac allografting. Untreated animals rejected allografts within a mean survival time (MST) of 7.2 +/- 0.8 days (n = 5). Injection (i.p.) of SPCs on both day -10 (n = 4) and day 0 (n = 6) induced significant prolongation of MST over the control (19.0 +/- 4.7, 14.2 +/- 2.1 days; p < 0.001 vs. control), while i.v. injection of SPCs on either day -10 (n = 4) or day 0 (n = 4) failed to do so (9.2 +/- 1.0, 8.5 +/- 0.6 days). Significant prolongation was still observed when irradiated SPCs were injected on day -10 (n = 4; MST: 19.0 +/- 5.4 days, p < 0.002 vs. control), but not when injected on day 0 (n = 5; MST: 9.4 +/- 2.1 days). These data suggest that the immunosuppressive effect of i.p. injection of donor SPCs could be induced by differential mechanisms according to the timing of inoculation of donor antigens.

Role of micro-chimerism in inducing immunological tolerance by intraportal injection of donor spleen cells in rats.

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) prevented liver allograft rejection. Moreover, we developed a new method using polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis, and demonstrated micro-chimerism (MC) at the DNA level in the spleen 14 days after IP injection. In the present study, the long-term presence of injected allogeneic SPCs was investigated at the cellular level by immunofluorescence staining as well as the DNA level using RFLP analysis. Male ACI (RT1a) rats were used as the donors and Lewis (RT1(1)) rats as the recipients. After DNA preparation from the lymphoid organs, RT1B beta domain 1 region was amplified by PCR, and RFLP analysis was performed with PvuII restriction enzyme. In the immunofluorescence staining, the monoclonal antibody, MN4-91-6, was used to detect the injected donor ACI SPCs in a frozen specimen. We did not detect MC in Lewis rats intravenously injected with 5 x 10(7) ACI SPCs on day 14. On the other hand, stable chimerism in the spleen was observed in intraportally injected rats up to 28 days after injection at not only the DNA level but also the cellular level. No chimerism was detected in other organs (including the thymus, lymph nodes, and liver). In conclusion, the long-term presence of injected allogeneic SPCs in the spleen was demonstrated after IP injection but not after IV injection, and this phenomenon may be one of the mechanisms involved in portal venous immunosuppression.

Efficacy of combination treatment--(TAE with adriamycin and ethanol)--for hepatocellular carcinoma.

Among 44 patients with hepatocellular carcinoma (HCC), combination treatment with both transhepatic arterial embolization (TAE) and ethanol injection therapy (EIT) was performed in 10 patients. Only two had tumors measuring less than 3 cm in diameter. In all, eight patients had solitary tumors and two had multiple tumors. The tumor was classified as stage I in one patient, stage II in six subjects, stage III in two patients, and stage IV in one subject prior to TAE, but one stage II case was changed to stage III after laparotomy. The clinical stage was I in two patients, II in six subjects and III in two patients. Five patients with tumors of stages I and II achieved either a complete response (CR) or partial response (PR). However, three patients with tumors of stages III and IV showed progressive disease (PD). Thus, the response rate (CR+PR) was 50%. For tumor stages I and II, the 1-, 2-, and 3-year survival values were 100%, 100%, and 83%, respectively. For tumor stages III and IV, the 1- and 2-year survival values were 75% and 25%, respectively. Combination treatment of HCC appears to be efficacious for tumor stages I and II.

New development of transarterial immunoembolization (TIE) for therapy of hepatocellular carcinoma with intrahepatic metastases.

The prognosis of patients with multiple hepatocellular carcinoma (HCC) remains disappointing. In this study, we devised a new therapeutic modality for HCC consisting of transarterial immunoembolization (TIE) using OK-432 and fibrinogen and then analyzed the preliminary results. In the first series, we applied the treatment to 19 patients with advanced HCC who had proved to be insensitive to several previous conventional treatments. In all, 14 patients (74%) with unresected HCC have currently survived for between 2 and 16 months after TIE. The remaining 5 patients died at 17, 14, 8, 7, and 4 months after TIE. The serum levels of tumor markers decreased in all of the patients, and a marked reduction in tumor size was observed in six patients after TIE. A high fever occurred in all cases, and abdominal pain and loss of appetite were also observed after TIE. However, deterioration of liver function was negligible. After confirmation of the safety of this method, we started a second study series in which this TIE treatment was selected as the first choice. Six patients have been treated to date. All patients in this group underwent hepatic resection at 6-48 days following TIE. Histological examination of the resected specimens following TIE showed massive infiltration of mononuclear cells around tumor cell nests and lytic necrosis as well as coagulation necrosis of the main tumor and the intrahepatic metastases. In conclusion, our results indicate that TIE may be a safe and promising therapy for patients with HCC.

Prognostic significance of c-erb B-2 protein status using an enzyme immunoassay in breast cancer.

Background. The prognostic significance of c-erb B-2 in breast cancer remains controversial. The aim of this study was to determine the practical prognostic significance of c-erb B-2 protein status in breast cancer extracts, using an enzyme immunoassay. Methods. An enzyme immunoassay was used to measure levels of c-erb B-2 protein prospectively in 360 patients with breast cancer, using cytosol fractions prepared for steroid receptor assay. The status of c-erb B-2 protein was assessed using a cut-off value for positivity of 18 ng/mg protein. Univariate and multivariate analyses were performed. To evaluate the prognostic significance of c-erb B-2 protein status. Results. Levels of c-erb B-2 protein in tumor tissue extract ranged from 0 to 213.0 ng/mg protein (mean, 15.5 ng/mg protein). In 52 tumors (14.4 %) more than 18.0 ng/mg protein was detected, and these tumors were regarded as c-erb B-2 protein-positive. Correlations were found between c-erb B-2 protein positivity and large tumor size (>3 cm; P = 0.0095), higher histological grade (P < 0.0001), estrogen receptor negativity (P < 0.0001), and progesterone receptor negativity (P < 0.0001). There was also a marginally significant correlation between c-erb B-2 protein positivity and lymph node positivity. Multivariate analysis showed that c-erb B-2 protein status was a significant independent prognostic factor for disease-free survival, being strongly significant in patients with positive lymph nodes. Conclusion. c-erb B-2-positive breast cancers are biologically more aggressive and c-erb B-2 protein status could be a candidate as a prognostic factor for patients with breast cancer, being particularly valuable in patients with positive lymph nodes.

Leiomyoma of the duodenum--a case report.

A 36-year-old man with anemia due to gastrointestinal bleeding, diagnosed to have a leiomyoma of the duodenum, is described. The conventional barium meal study and gastroendoscopy were useful for diagnosing the lesion. More invasive radiological methods, such as abdominal angiography and technetium-99m scintigraphy failed to detect the tumor. Early diagnosis and resection of the tumor would result in a good prognosis.

Carcinoma of the head of the pancreas after excision of a choledochal cyst.

We here report a 53-year-old woman who had undergone resection of a choledochal cyst and hepaticojejunostomy three years before. She was readmitted because of intermittent fever, and abdominal computed tomography revealed a 4-cm tumor in the head of the pancreas. We performed pancreatoduodenectomy, and examination of the resected specimen showed well-differentiated papillary adenocarcinoma. Only 5 cases of carcinoma occurring after the resection of a choledochal cyst have been reported, and to our knowledge, this is the second case of carcinoma of the head of the pancreas.

Surgical treatment of hepatic caudate lobe metastases originating from colorectal primaries.

Malignant tumor resection of the hepatic caudate lobe has recently received attention. However, there are few reports about metastatic liver tumor in the caudate lobe from colorectal carcinoma, and its clinical features still remain unknown. In this paper, three patients operated on in our institute and 15 reported cases from the published literature were analyzed in order to reveal clinical features of this disease. Many cases had advanced liver tumors, such as invasion in to major vessels at the time of operation. Isolated complete caudate lobectomy was performed in 8 patients and major hepatectomy was carried out in 6 instances. Seven cases also underwent partial resection of the inferior vena cava. Recurrence of disease was observed in 11 patients: seven cases had relapse only in the residual liver, five of whom underwent another hepatectomy. The median survival time of those patients who died was 25 months, and that of seven cases with IVC resection, 18 months. Two patients out of five who received a second hepatectomy survived for longer than 90 months. It is suggested that aggressive surgical treatment including repeated hepatectomy results in the prolongation of survival. Earlier diagnosis and surgical treatment at a more appropriate stage of the disease may further improve the survival rate.

[Paradoxical effect of L-arginine on nitric oxide (NO) synthesis and histopathological changes in 5/6 nephrectomized SD rats].

Whether L-Arginine (L-ARG) ameliorates or aggravates renal function and histopathological changes in several models of renal disease remains controversial as L-ARG is the substrate for nitric oxide (NO) synthase as well as the precursor of proline and polyamines which cause renal fibrosis. These ambiguous results might be attributed to differences in the dose and period of L-ARG administration and the animal model used in each observation. Therefore, we tested the dose-dependent effect of L-ARG on mean blood pressure (MBP), 24-hour urinary excretion of protein (UP), NO metabolites (NO2(-) + NO3-) and cyclic GMP (cGMP), plasma asymmetrical dimethylarginine (ADMA), glomerular sclerosis index (SI) and % interstitial fibrosis area (%INT) in 5/6 nephrectomized SD rats. These 5/6 nephrectomized SD rats were divided into 4 groups: 1. L-ARG 0.2 g/kg/day (0.2 g ARG), 2. L-ARG 1 g/kg/day (1 g ARG), 3. L-ARG 2 g/kg/day (2 g ARG), 4. No administration of L-ARG(ARG(-)). Compared with ARG(-)MBP, UP and ADMA were significantly decreased and NO2(-) + NO3-, cGMP were significantly increased in the 0.2 g ARG. SI group and %INT were significantly increased in the 2 g ARG group and decreased in the 0.2 g ARG group. A small dose of L-ARG ameliorated glomerulosclerosis and interstitial fibrosis while a larger dose did not. SI, %INT and ADMA were inversely correlated with NO2(-) + NO3-. These data suggested that renal NO synthesis might attenuate glomerulosclerosis and interstitial fibrosis and the rise in ADMA and L-ARG might cause the decrease in NO.

[A case of hepatocellular carcinoma responding to hepatic arterial infusion chemotherapy with low-dose cisplatin and 5-fluorouracil].

A 74-year-old man had multiple liver recurrence of hepatocellular carcinoma (HCC) after extended left hepatectomy. He was treated by continuous hepatic arterial infusion (HAI) chemotherapy with low-dose cisplatin (CDDP) and 5-fluorouracil (5-FU) via an implanted reservoir. A catheter was inserted percutaneously into the hepatic artery using the Seldinger technique. The patient was administered 10 mg of CDDP on day 1 and 500 mg/day of 5-FU for 4 days as one course. Four courses were administered and the PIVKA-II level decreased from 427 to 216 mAU/ml. However, infusion port problems led to interruption of chemotherapy and PIVKA-II increased to 798 mAU/ml. His chemotherapy was changed to 10 mg of CDDP on day 1 and 750 mg/day of 5-FU for 2 days. After five courses were administered, PIVKA-II decreased to 540 mAU/ml. This patient is still alive 15 months after the start of therapy. This case suggests that HAI with low-dose CDDP and 5-FU might be useful for prolonging the survival of HCC patients with a good quality of life.

[Case report--efficacy of short-term intraarterial 5-fluorouracil for liver metastasis from colorectal cancer].

Hepatectomy has the highest cure rate among the various methods for treating liver metastasis from colorectal cancer. We previously reported that continuous hepatic arterial infusion (HAI) of 5-FU is effective for improving the prognosis of patients with liver metastasis. In this study, we examined the efficacy of short-term continuous HAI of 5-FU for treating liver metastasis from colorectal cancer. A 57-year-old woman with a solitary liver metastasis from rectal cancer was treated by continuous HAI of 5-FU (1,000 mg/day) for 6 days. Her elevated serum CEA level (20.7 ng/ml) then returned to normal. Computed tomography revealed a decrease of 55.6% in the size of the liver tumor. Partial segmentectomy (S7) was subsequently performed. Histological examination of the resected tumor showed marked degeneration, necrosis, fibrosis, and calcification with viable moderately differentiated adenocarcinoma cells. These results suggest that preoperative HAI of 5-FU is safe and worth trying in patients with liver metastasis from colorectal cancer.

[Microwave coagulation therapy for liver recurrence after resection of metastatic colorectal cancer--comparison with re-hepatectomy].

One hundred and fifty-three patients with liver metastases from colorectal cancer underwent hepatectomy from 1979 to 1998. Recurrence in the residual liver occurred in 71 of the 129 patients with curative B resection, and re-hepatectomy has been done in 21 of these 71 patients since 1984. The 5-year survival rate is 37.0%. MCT has been performed in ten of these 71 patients who could not undergo re-hepatectomy and one patient received both therapies. The 3-year survival rate is 66.7%, which is statistically better than that of 35 patients who could not undergo re-hepatectomy before we started MCT (3-year survival rate of 20.0%). Re-hepatectomy is quite effective for recurrent liver remetastasis after hepatectomy, and MCT is probably of similar value.

[Efficacy of continuous hepatic arterial infusion of high-dose 5-FU for liver metastases of gastric cancer].

We studied the efficacy of continuous hepatic arterial infusion of high-dose 5-FU (high-dose CHAI) in two patients with multiple (five or more) bilobar liver metastases of gastric cancer. 5-FU was given continuously via the hepatic artery at 1 g/day for 3 days, followed by one day off therapy and repetition of the initial treatment as one course. Case 1 was a 67-year-old man with Borrmann type 2 gastric cancer who had undergone total gastrectomy. Metachronous multiple liver metastases (maximum diameter: 3.5 cm) were detected at 11 months after surgery. One month later, we started high-dose CHAI and gave two courses with a 4-day interval between them. After that, 5-FU was given twice by hepatic arterial infusion (HAI) at dose of 1.5 g/week. The tumor diameter had decreased by 50% at 3 months after high-dose CHAI. Case 2 was a 64-year-old man with Borrmann type 3 gastric cancer who had synchronous multiple liver metastases (maximum diameter: 9 cm) and liver dysfunction. One month after distal gastrectomy, we started high-dose CHAI and finished one course. After that, liver function returned to normal and 5-FU was given by HAI at dose of 1 g/week on an outpatient basis. The tumor diameter decreased to 1/3 of the initial size at four months after high-dose CHAI. High-dose CHAI using 5-FU may be safe and effective for liver metastases from gastric cancer.

[Evaluation of prophylactic hepatic arterial infusion chemotherapy after hepatectomy for metastases from colorectal cancer--the second report].

We followed 18 patients who underwent curative hepatectomy for metastatic carcinoma of the colon from March 1993 to March 1995, and investigated their survival and the effect of treatment on recurrence. The patients were randomly divided into two groups. Group A (n = 9) was given continuous 5-FU (500 mg x 4 days/week) for six weeks from 2 weeks after surgery via the hepatic artery and Group B (n = 9) was given 5-FU orally from 2 weeks after surgery. The cumulative one-, two-, and three-year survival was 88.9, 88.9, and 76.2% in Group A, while the one- and two-year survival was 100 and 80% in Group B. The one-, two-, and three-year disease-free survival was 77.8% in Group A, while the one- and two-year disease-free survival was 55.6 and 29.6% in Group B (p = 0.0369: Mantel-Cox). These findings suggest that continuous hepatic artery infusion of 5-FU is effective against post-hepatectomy recurrence of metastatic carcinoma of the colon.

[Features of colorectal cancer with fecal-occult-blood tests--comparison with colorectal cancer with no screening].

BACKGROUND: There is now evidence from three randomized controlled trials (from Minnesota, Nottingham and Funen) that screening average-risk individuals for colorectal cancer (CRC) with fecal-occult-blood tests (FOBT) can reduce mortality from CRC. In Japan, mortality rates from CRC have increased and mass screening with FOBT has been performed since 1992. Although there is growing proportion of CRC with FOBT, there is no conclusive evidence that they reduce mortality from this cause. We evaluated the feature of CRC with FOBT, as one of the methods for evaluation of the efficacy of screening with this test. METHODS: Between January, 1982 and December, 1996, 2071 cases with CRC resected in our hospital were considered. We evaluated the clinicopathological findings about age, sex, location of tumor, size depth, incidence of lymph node metastasis, stage and prognosis of CRC with FOBT (376) compared with CRC with no screening (controls; 1695). RESULTS: CRC with FOBT were earlier stage and smaller-sized cancers than controls. No significant difference was found in the incidence of lymph node metastasis at each depth and prognosis in each stage. CONCLUSIONS: By screening with FOBT, we can detect at an earlier stage and with a smaller cancer, with the same biological behavior as controls. Now, we must encourage colon screening, continuing to research ways to improve identification of high-risk subgroups and increase complicance.