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We report herein the case of a 67-year-old man who was admitted to our hospital with a 3-month history of hoarseness, a cervical mass, and weak muscles of the extremities 6 years after undergoing a right colectomy for carcinoma. Physical and imaging examinations disclosed a nodule in the thyroid with swollen cervical lymph nodes, multiple pulmonary lesions with pleural effusion, and a cerebral mass. Fine-needle aspiration cytology of the thyroid nodule and brush cytology of the lung mass revealed adenocarcinoma, which was consistent with a diagnosis of metastases from the primary colon adenocarcinoma to the thyroid, brain, and lung. The patient eventually died from the malignancy, although he survived for 4 months after the disclosure of the metastases. The rarity, diagnosis, and prognosis of thyroid metastasis from colon carcinoma are discussed.

A 67-year-old male complaining of constipation with a change in stool caliber for several months visited our hospital in June 1999. A positive test for occult blood in the feces led to the disclosure of a type II carcinoma of the sigmoid colon with multiple liver metastases. A lymph node dissection with a sigmoidectomy disclosed no metastases histologically, so a left hepatectomy and enucleations of the metastases were performed. In addition, the invaded middle hepatic vein (MHV) was resected and repaired using a peritoneal patch. The patient's postoperative course was uneventful until July 2000, when computed tomography of the liver showed a single nodule measuring 3 cm in diameter in segment 6. The metastasis was excised in August. Since then, the patient has shown normal tumor marker values. The MHV has remained patent for 24 months after its reconstruction. A resection of the liver metastases including venous reconstruction is beneficial for patients since it results in a longer survival and allows for venous drainage of the residual liver. The peritoneum is also accessible, enabling the fitting of a patch graft for hepatic vein repair.

This is a report of a case of liver cell adenoma (LCA) in a 26-year-old man with no prior history of liver disease or glycogen storage disease and no record of hormonal therapy. He was found to have an asymptomatic hepatic mass during a routine medical examination. The physical findings were unremarkable, and the results of routine laboratory studies were all within normal limits. Selective hepatic arteriography showed a hypervascular mass within the right lobe of the liver. Despite the radiological examination, the nature of the mass was unknown, and preoperative biopsy was unadvisable because of the risk of bleeding. Because of the difficulty of determining the malignancy of the hepatic tumor preoperatively, elective laparotomy for diagnosis and hepatectomy as treatment appeared to be the best available approach. Pathological examination of the surgical specimen resulted in a diagnosis of LCA. A review of the literature revealed that LCA unassociated with the use of oral contraceptives is rare.

In a 63-year-old woman computed tomography (CT) incidentally detected a celiac artery aneurysm approximately 3 cm in diameter. While conventional angiography suggested that the splenic artery and common hepatic artery arose from the celiac artery aneurysm, three-dimensional CT angiography indicated that the aneurysm involved only the mid portion of the celiac artery. Considering the risk of eventual aneurysm rupture, surgery was performed. Aneurysmectomy and devascularization of hepatic, splenic, and celiac arteries were carried out following complete cross-clamping of the celiac artery. The distal segment of the celiac artery was directly anastomosed to the proximal segment in an end-to-end fashion. Histologically, the aneurysm wall showed atheromatous changes. Contrast-enhanced abdominal CT confirmed complete removal of the celiac artery aneurysm, and postoperative angiography confirmed good arterial flow. The patient recovered uneventfully after surgery, with normalization of transiently abnormal hepatic function parameters. In this case of celiac artery aneurysm, three-dimensional CT angiography was found to be valuable in determining the relationships of the aneurysms to important arterial branches.

Routine transplant aspiration cytology (TAC) after liver transplantation gives detailed information that concerns immunologic events in the graft. TAC can be helpful for diagnosis of acute rejection, but it also detects morphological signs of rejection without clinical correlate ("subclinical rejection"). The aim of this study was to systematically evaluate factors that influence the development of early clinical and subclinical rejection and to analyze the relevance of these early immunologic processes for the long-term course. The study includes the course of 340 patients after liver transplantation between 1988 and 1995 in whom TAC was performed routinely and who were followed for a minimum of 3 years. TAC findings were correlated with the following various clinical parameters: (1) Overall early clinical rejection occurred in 17.4%, subclinical rejection in 59.1%, and no immune activation was seen in 23.5% of patients. (2) Incidence of early clinical and subclinical rejection was markedly influenced by type of immunosuppression. (3) Basic disease and extent of preservation injury had only a minor influence; there was a trend towards lower early rejection associated with more severe preservation damage, increased patient age, and early retransplantation. (4) Presence of early clinical or subclinical rejection was not associated with a higher incidence of chronic dysfunction. (5) Falsely indicated antirejection treatment was associated with inferior graft survival. Subclinical rejection is very frequent early after liver transplantation, requires no treatment, and has no long-term adverse effect. Incidence of early clinical rejection is mainly determined by initial immunosuppression; its occurrence has no negative long-term effects and may even be associated with a lower risk for later immunological complications. Thus, the incidence of early acute rejection is no adequate parameter for evaluating the quality of an immunosuppressive treatment protocol.

Background/aims: We encountered a case of posthepatectomy splenic enlargement and hypersplenism followed by disseminated intravascular coagulopathy with airway hemorrhage causing death., Methodology: We, therefore, retrospectively investigated postoperative splenic enlargement, hypersplenism and disseminated intravascular coagulopathy by computed tomography and laboratory data in 57 hepatectomized patients with a malignant or benign disease in the postoperative period., Results: Of 32 patients with hepatocellular carcinoma or biliary tract carcinoma (group A), 12 with metastatic hepatic lesions (group B), and 13 with benign liver disease (group C); remarkable (20%) splenic enlargement was noted in 8 patients in group A, 2 in group B, and 2 in group C. Seven of the 12 patients were associated with liver cirrhosis, 5 with preoperative splenomegaly, and 8 had undergone major hepatectomy. Postoperative hypersplenism developed in 5 patients in group A, and one patient in group C. All of them were associated with liver cirrhosis or chronic hepatitis and preoperative splenomegaly, and five had undergone hepatic lobectomy or more extensive resections. All except for the disseminated intravascular coagulopathy case recovered. Statistically, splenic enlargement was significantly related to the extent of hepatectomy; lobectomy versus segmentectomy = 28.3 +/- 28.5% (n = 14) versus 12.4 +/- 13.8% (n = 20), (unpaired Student's t test, P = 0.037). Platelet counts of the patients with liver cirrhosis or chronic hepatitis is lower than those without the diseases, both pre- and postoperatively (14.0 +/- 6.0 x 10(4)/mm3 vs. 21.5 +/- 6.2 x 10(4)/mm3, P = 0.0001)., Conclusions: Postoperative hypersplenism was noted only in the patients with liver cirrhosis or chronic hepatitis and preoperative splenomegaly, and developed more frequently after larger hepatectomies than after smaller hepatectomies; 5 (45%) of 11 versus 1 (7%) of 14, chi 2 test, P = 0.026).

A 39-year-old man hospitalized with upper abdominal pain had been found to have a 3mm polyp in the body of the gallbladder 3 years previously. Laboratory tests on admission showed mild liver dysfunction. Ultrasonography depicted a dilated gallbladder with increased wall thickness; the polyp could no longer be seen. Computed tomography with drip infusion cholangiography again showed a dilated gallbladder, and also stenosis of the distal cystic duct. The resected specimen obtained by laparoscopic cholecystectomy showed disappearance of the polyp from the body of the gallbladder. A cholesterol stone was incarcerated in the cystic duct, representing an impacted detached cholesterol polyp causing acute cholecystitis. Spontaneous detachment of a cholesterol polyp from the gallbladder mucosa, then, can result in acute cholecystitis.

The spontaneous disappearance of a hepatic cyst is described. A 62-year-old woman presented with symptoms of general fatigue in August 1992. Her past medical history was significant for chronic hepatitis, which was diagnosed in 1990 but not treated. Initial laboratory tests revealed mild liver dysfunction with a positive serologic test for hepatitis C. In August 1992, ultrasonography and computed tomography disclosed a cystic lesion along the middle hepatic vein in the right anterior segment of the liver, which was 40 mm in diameter. Repeat radiologic studies in June 1994 demonstrated that the size of the cyst was unchanged. In May 1995, the cyst was only 25 mm in diameter, and it continued to decrease in size thereafter, to 10 mm in September 1995 and 7 mm in September 1996. No hepatic cyst was visualized in December 1996, though the region in which the cyst existed was hyperechoic. Laboratory data were essentially unchanged from August 1992 to December 1996. Clinically the patient remained asymptomatic.

Liver transplant aspiration cytology(TAC) contributes significantly to the differential diagnosis of transplanted liver graft dysfunction, since each of the possible causes induces characteristic cytological findings in the graft and peripheral blood. That is, TAC can differentiate various inflammatory changes as indicating acute rejection, viral infection, or bacterial infection. The key is the intensity of activated lymphocytes in the graft and in blood. Remarkable lymphocytic activation only in the liver graft indicates acute rejection, meanwhile in a case of viral infection the activation can be seen both in liver graft and blood. The patterns and degree of liver cell injury and cholestasis can also identify the reason for liver impairment. Several cases of acute rejection, viral infection, and subclinical acute rejection diagnosed by TAC were recognized on TAC slide preparations. Subclinical rejection, which means acute rejection appreciated only cytologically or histologically, can be followed without treatment unless the patient demonstrates jaundice and liver enzymes over 100 units. In a case of chronic rejection, TAC is not an appropriate method of diagnosis, because there are few infiltrating cells in the portal triad, and subsequently, no visible activated lymphocytes on TAC slides. Although TAC is not well accepted, it is an informative means preliminary to core biopsy, and its use is expected to become wide-spread in the future because it is a safe procedure with minimal invasiveness to patients, and useful for differential diagnosis. The use of TAC will lessen the frequency of performing more invasive core biopsy.

Purpose: Soluble Fas antigen (sFas) and ligand (sFasL), which are associated with apoptosis, have not been evaluated in gastrointestinal or cerebrovascular surgery. The aim of this study was to measure these substances in serum, intra-abdominal fluid, or cerebrospinal fluid, and to speculate a pathophysiologic role for Fas-FasL apoptosis in surgery., Materials and Methods: Arterial blood and intraabdominal or cerebrospinal fluid were collected at intervals from 27 gastrointestinal surgical patients, 10 cerebrovascular surgical patients, and 10 spinal anesthesia patients., Results: Serum sFas levels did not change during and after surgery. Intra-abdominal and cerebrospinal sFas levels were identical to and lower than those in serum. Serum sFasL levels did not change during surgery, but decreased after surgery. Intra-abdominal and cerebrospinal sFasL levels were higher than and identical to those in serum. In spinal anesthesia, cerebrospinal sFas and sFasL levels were lower than those in serum., Conclusions: Serum sFasL decreases after surgery, whereas intra-abdominal or cerebrospinal sFasL increases, although sFas in each site does not change, suggesting redistribution of activated lymphocytes into local surgical wounds and induction of apoptosis in this site.

We were able to isolate a lysosomal fraction from rat brain which had a higher degree of purity than in previous studies with good recovery. This has been made possible by using percoll gradients following the swelling of mitochondria in the presence of calcium which would eliminate contamination from small amounts of mitochondria. By using percoll density gradient centrifugation after the swelling of mitochondria in the presence of calcium, cerebral lysosomes were purified 312-fold with the recovery of approximately 22 %, which is the highest reported for any cerebral lysosomal preparation. The most effective procedure for the separation was achieved by using 1.25 mM calcium incubation with post nuclear fraction. As brain lysosomes may play a major role not only in degrading macromolecules but also in their transport to the deposition site, obtaining purified rat cerebral lysosomes represents an important step in the study of the generation of macromolecules which accumulate in the brain.

We present a new intraarterial infusion chemotherapy technique for the treatment of inoperable cancer in the body and tail of the pancreas. The spleen was embolized at its hilum with coils to infuse an anti-tumor agent selectively into the pancreatic parenchyma and a catheter was placed into the splenic artery and connected to the reservoir. 99mTc-macroaggregated albumin+diethylenetriaminepenta-acetic acid (MAA+DTPA) was injected into the reservoir and the body and tail of the pancreas were visualized on the image. For inoperable cancer in the body and tail of the pancreas, splenic artery embolization followed by the placement of a catheter into the splenic artery can deliver a highly concentrated anti-tumor agent to the tumor.

The A and J series of prostaglandins (PGs) accumulate in the nuclei to suppress the proliferation of cancer cells. Here we report that Delta7-PGA1 methyl ester, a synthetic anti-cancer PG, increased the level of mRNA for the cyclin-dependent kinase inhibitor p21 in human leukaemia HL-60 cells. The induction of p21 was associated with the accumulation of hypophosphorylated retinoblastoma protein (pRB) and the suppression of c-myc gene expression. Since the p53 gene is deleted in HL-60 cells, the anti-cancer PG is suggested to inhibit cancer cell growth by inducing p21 via a p53-independent pathway. Unlike HL-60 cells, cisplatin-resistant HL-60/R-CP cells were insensitive to Delta7-PGA1 methyl ester. While c-myc expression was transiently suppressed, neither G1 arrest nor hypophosphorylation of pRB was observed with the anti-cancer PG. Plasma membrane vesicles from HL-60/R-CP cells showed an enhanced level of GS-X pump (ATP-dependent glutathione S-conjugate export pump) activity towards the glutathione S-conjugate of Delta7-PGA1 methyl ester (Km 110 nM). GIF-0019 ¿N-carbomethoxy-S-[5-(4-benzoylphenyl)pentyl]glutathione dimethyl ester¿, a specific inhibitor of the GS-X pump, dose-dependently enhanced the cellular sensitivity of HL-60/R-CP cells to Delta7-PGA1 methyl ester and induced G1 arrest. The GS-X pump is suggested to play a pivotal role in modulating the biological action of the anti-cancer PG.

It is known that some organophosphates produce not only well-known acute toxicity but also characteristic delayed neurotoxicity. Tri-ortho-tolyl phophate (TOTP), which was formerly named Tri-ortho-cresyl phosphaete (TOCP), was first noticed in an incident of poisoning as the compound which produced organophosphate induced delayed neurotoxicity (OPIDN). It is said that triphenyl phosphite (TPP) is also one of the organophosphates which possesses OPIDN. However, it is thought that TPP-induced delayed neurotoxicity (TPP-DN) is not identical with classical OPIDN. An intermediate syndrome was later proposed as the third neurotoxicity caused by organophosphates. We think that TPP is a model chemical of the third neurotoxicity. We compared TOTP with TPP using Japanese quails. We measured cholinesterase (ChE) activity and clearly demonstrated the difference between the two chemicals, that is to say, the activity recovered after 72 hrs from the administration of TPP, whereas the inhibition continued for more than 11 days after the administration of TOTP.

To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to be 5'-end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly2 in the myristoylation signal with Ala entirely inhibited the incorporation of [3H]-myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro translation/transcription system.

We provide evidence that the expression of the human MRP/GS-X pump encoded by the MRP (multidrug resistance associated protein) gene is induced by cisplatin in human leukemia HL-60/R-CP (cisplatin-resistant) cells and modulates cell growth inhibition by delta(7)-prostaglandin A1 (PGA1) methyl ester. The MRP mRNA level in HL-60/R-CP cells increased remarkably after a 24-h incubation with 20 microM cisplatin; interestingly, however, no amplification of the MRP gene was detected. In cisplatin-sensitive HL-60 cells, which express the MRP/GS-X pump at low levels, c-myc expression was substantially suppressed by delta(7)-PGA1 methyl ester and the cell cycle was arrested in G1 phase. By contrast, in HL-60/R-CP cells overexpressing the MRP/GS-X pump, c-myc expression and cell proliferation were much less affected by delta(7)-PGA1 methyl ester. This suggests that induction of the MRP/GS-X pump may confer on cancer cells resistance to anticancer prostaglandins and that the resistance mechanism may involve the increased efflux of PG-glutathione conjugates, as active intermediates, from the cells via the MRP/GS-X pump.

The tumor necrosis factor (TNF) mutant TNF-SAM2 has previously been shown to have a therapeutic profile superior to parental TNF. To initially evaluate the characteristics of liposomal formulations of TNF-SAM2, it was modified with the N-hydroxysuccinimide ester of caprylic acid to increase its hydrophobic binding to multilamellar and small unilamellar vesicles (MLVs and SUVs). Native PAGE and fluorescamine analysis of acetylated parental TNF and TNF-SAM2 indicated that these proteins both displayed trimeric structures based on crosslinking/SDS-PAGE analysis and behaved similarly with respect to reactivity of their amino functions. Limited N-terminal sequencing analysis of partially acetylated (approx 3 acetyl groups per trimer) TNF-SAM2 indicated that the N-terminal Val was not modified; this was also concluded based on HPLC/mass spectrometric (LC-MS) analysis of Glu C digests. LC-MS analysis of tryptic digests of the acetylated TNF-SAM2 indicated that Lys-98 was unreactive. Molecular ions corresponding to acetylated Lys-containing peptides for all five other Lys residues could be detected; none appeared hyperreactive, but Lys-11 appeared hyporeactive. MLVs composed of DMPC/DMPG (7:3) and SUVs composed of DPPC/DSPC (1:1) displayed high capacity for binding to caprylated TNF-SAM2. These formulations of caprylated TNF-SAM2 displayed tumor necrotizing and growth-inhibitory activity in a syngeneic tumor model, and may be candidates for clinical development.

We have evaluated the hemodynamic effects of systemically administered recombinant human tumor necrosis factor (TNF)-alpha, TNF-SAM2 and liposome-bound TNF-SAM2 in an anesthetized mongrel dog model. A dose of 10 micrograms TNF protein/kg of each formulation was injected in a peripheral vein and mean systemic arterial pressure (SAP), heart rate (HR) and cardiac output (CO) were measured. TNF-alpha induced a marked drop in SAP in all three dogs (mean decrease = 59.3 +/- 5.2 mm Hg; to 61.5% of baseline; p = 0.008); whereas TNF-SAM2 caused a smaller and transient drop in SAP in four dogs (mean decrease = 25.5 +/- 10.1 mm Hg; to 81.2% of baseline; p = 0.086). In three dogs administered liposome-bound TNF-SAM2, which retains antitumor activity in vivo, a net slight hypertensive phase and sustained elevated CO occurred, followed by a return to an essentially normotensive state (101.0% of baseline SAP). This model demonstrates that the principal acute systemic toxicity of TNF, i.e., hypotension, can be markedly attenuated by liposomal formulation of a second-generation TNF.

Sixty-one Japanese white rabbits underwent total thymectomies. Eight of the animals died of intraoperative hemorrhage or respiratory failure, and twelve died of postoperative hypovolemia, dehydration, malnutrition or wound infections. Forty-one rabbits survived and were used as a total thymectomy model. The whole procedure of our technique is described in detail so that even those with no experience of performing thymectomies can follow it. Some tactics especially how to deal with the thymic vessels and the pleurae, are key points. Our technique permits safe and perfect total thymectomies to be carried out on rabbits.

Neuroblastic tumor cases in our departments were evaluated in terms of the stage of the tumor, N-myc amplification, urinary vanillylamndelic acid (VMA)/homovanillic acid (HVA) and survival rate. Two asymptomatic cases, diagnosed when under a year old by mass screening, had no amplifications of N-myc but showed more than one value of urinary VMA/HVA ratio. The patients are now doing well eight years after complete excision of the neuroblastoma which had originated in the sympathetic ganglion. On the other hand, two other symptomatic cases, operated on at the ages of 3 and 5 years, showed remarkable amplifications with less than one value of urinary VMA/HVA, and died from the tumor soon after partial resection of the neuroblastoma and ganglioneuroblastoma which had originated in the adrenal gland and the sympathetic ganglion, respectively. The present monograph reports our cases and discusses prognostic factors.

A 67 year old male with non-resectable hepatocellular carcinoma (HCC) in both lobes and liver cirrhosis was treated with transcatheter arterial embolization and regional chemotherapy. He was doing well for 18 months. He was readmitted for fever, chest pain and multiple pulmonary metastases. During interleukin-2 therapy, he suddenly developed dyspnoea and palpitation, and was in shock. Left-sided haemothorax was confirmed by draining 3 L of fresh blood. In spite of intensive care, he died within 36 h. Autopsy showed that the haemothorax was caused by rupture of one of the metastases in the upper lobe of the left lung, and that the primary HCC was totally necrotic. Survey of the literature failed to find a report of fatal bleeding from a lung metastasis of HCC.

A forty-nine-year-old female patient, complaining of swallowing difficulties and general fatigue, was admitted to the first hospital of Nippon Medical School. At the age of 32, she was operated on for the removal of a well encapsulated non-invasive thymoma. Since then, she had been well till the age of 46, when chest X-ray films showed a recurrent thymoma which was excised together with the complete thymic tissues. One year later, she developed myasthenia gravis (MG) with a ptotic right upper eyelid and general fatigue. Subsequently, she was placed on medication. After 21 months, however, she died of myasthenic crisis in spite of vigorous respiratory and nutritional support. The autopsy revealed a small residual thymoma on the left lung, and systemic atrophy of the skeletal muscles. In this paper, the mechanism of post-thymectomy MG and the recurrence of non-invasive thymoma are discussed.

One hundred laparoscopic cholecystectomies were performed since April 1991. Eleven patients were treated with a new technique without CO2 insufflation, using a traction device to elevate the right upper quadrant wall. Two Kirschner wires were introduced subcutaneously to permit the abdominal wall to be lifted for satisfactory laparoscopic view, as the gas insufflation technique yields. Preoperative evacuation of the intestines and intraoperative muscle relaxation are necessary for easy and successful cholecystectomy. Three cases were converted to laparotomy because of remarkably distended intestine due to incorrect endotracheal intubations. No complications related to subcutaneous wire traction technique were noted in this series. Subcutaneous wire traction technique provides a simpler, and possibly safer alternative to the gas insufflation technique.

Reconstruction following total gastrectomy usually requires one of the various methods of esophagojejunal anastomosis. We present herein the case of a 78-year-old woman with gastric carcinoma involving the adjacent organs for which resection of the whole stomach, distal pancreas, transverse colon, and distal duodenum was performed. Reconstruction was carried out with an esophagojejunostomy, followed by a side-to-side anastomosis between the raised jejunal limb and the residual short duodenum. Her postoperative course was uneventful and she was able to lead a relatively comfortable life at home until she died after tumor recurrence caused GI bleeding 1 year postoperatively. This new modified Roux-en-Y with an ultra-short duodenum can be applied for such contiguously invasive gastric carcinoma to achieve a better quality of life, when conventional reconstruction techniques after total gastrectomy are not indicated.

To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.

The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.

When stimulated with different stimuli, neutrophils generate various active oxygen species. These active oxygen molecules can be analyzed by luminol chemiluminescence (LCL). Phosphatidylserine (PS)-liposomes increased the formylmethionyl-leucyl-phenylalanine-induced LCL of guinea pig peritoneal neutrophils without affecting their oxygen consumption and superoxide (O2.-) generation. Similar effects of PS-liposomes were also observed in LCL of neutrophils stimulated by phorbol myristate acetate or arachidonic acid but not by opsonized zymosan. Kinetic analysis revealed that the PS-liposome-induced increase in LCL depended on extracellulary generated O2.-. Moreover, the stimulatory effect of PS could be seen only when it formed liposomal membranes. The effect of PS-liposomes was also inhibited by superoxide dismutase, catalase, and deferoxamine, an iron chelator, but not by azide, an inhibitor of myeloperoxidase. Similar enhancement of stimulation-dependent LCL response was also observed with Fe3+ and ADP-Fe3+, but the degree of enhancement was much greater with PS-liposomes than with iron and its complex. The increase in hydroxyl radical generation by PS-liposome-treated neutrophils was confirmed by experiments with EPR spectrometry using spin-trapping agents. These results suggested that the interaction of neutrophils with PS-containing membrane surface might generate reactive oxygen species that enhance the stimulus-dependent LCL response of neutrophils.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.

The mechanism by which nonsteroidal antiestrogen inhibits Ca(2+)- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.

Evaluation of cellular immunity against human melanoma was performed in an in vivo model using Balb/c nude mice bearing the human melanoma. Intraperitoneal injection of 1 X 10(7) melanoma cells produced peritonitis carcinomatosa which lead to death of the mice at 23.8 +/- 2.6 days (N = 12). Peripheral blood lymphocytes (PBL) from healthy donors and the melanoma cells cultured on the back of the nude mouse were given to nude mice intraperitoneally, and survival times of 22.0 +/- 2.3 days were observed (N = 8). PBL from four of the five healthy donors which have been presensitized on monolayers of the melanoma tissue culture cells in flask failed to prolong host survival times. In contrast, PBL from 15 of 20 melanoma patients were found to prolong the survival of the tumor bearing nude mice. Of these 15 patients, 8 were treated with specific active immunotherapy, but 7 had not been sensitized except by the disease process. The ability of PBL from patients to prolong survival of the melanoma bearing nude mice did not seem to correlate with either the stage of the disease or the clinical course. The possible mechanisms for the prolonged survival and the usefulness of this in vivo model were discussed.

Evaluation of cell mediated immunity against human melanoma target cells was performed in an in vivo model using human tumor xenografts growing in Balb/c athymic mice. Intraperitoneal inoculation of 1 x 10(7) human melanoma cells produced carcinomatosis which lead to death of the animals at 23.8 +/- 2.6 days (N = 12). Peripheral blood lymphocytes (PBL) from normal donors were administered to tumor bearing mice, and survival times of 22.0 +/- 2.3 days were observed (N = 8). Peripheral blood lymphocytes from the four of five normal donors which had been presensitized on monolayers of melanoma tissue culture cells in vitro failed to prolong host survival times. In contrast, PBL obtained from 15 or 20 melanoma patients were found to prolong survival of the tumor bearing nude mice. Of these 15 patients, 8 were undergoing specific active immunotherapy, while 7 had not been sensitized except by the disease process. The ability of PBL obtained from patients to prolong survival of tumor bearing animals did not appear to correlate with either the stage of the disease or the patient's clinical course. The possible mechanisms for the prolonged survival and usefulness of this model are discussed.

Mouse hepatitis virus (MHV) infection was found in 15 of 40 athymic mice (Balb/c nu/nu) used in an experimental model designed to study the effect of cell mediated immunity on target human melanoma cells growing in the peritoneal cavity. Most non-MHV-infected mice died of peritoneal tumor within predictable periods following injection of the melanoma cells. Twelve of 15 MHV-infected mice which showed either poor or no tumor growth died of MHV infection, whereas only 3 showed good tumor growth. Peritoneal macrophages in the ascites from the MHV-infected mice were greatly reduced in number compared to those in the ascites from the non-MHV-infected mice. Although the fibrohistiocytic reaction was more prominent in the MHV-infected mice, other aspects of spleen and lymph node lymphocytic reactions exhibited no remarkable differences between the two groups. The possible mechanisms whereby MHV infection influences tumor growth in nude mice are discussed.

A subcutaneous portal reservoir system has been developed for infusing chemotherapeutic agents intraportally to treat nonresectable metastasis to the liver. We have discussed the clinical use and effectiveness of the system.

Intraperitoneal injections of 2 X 10(7) SH-Me cells (human metastatic melanoma cells)( to 20 Balb/c nu/nu mice (Group A) and 1 X 10(7) cells to 20 mice (Group B) were performed. All animals were studied clinicopathologically. Five animals in Group A were sacrificed serially, revealing marked tumor growth of the melanoma within the peritoneal cavity. These tumors grew in multiple nodular configurations and tumor ascites was present by the third week. The remaining 15 animals in Group A were allowed to progress and seven subsequently died with mouse viral hepatitis (MVH). These animals had suppressed tumor growth. The remaining eight animals died of peritoneal carcinomatosis with survival time of 24.1 +/- 5.0 days. Eight of the animals in Group B died of mouse viral hepatitis while the remainder died of peritoneal tumor without distant metastasis. Survival time in these animals was 23.8 +/- 2.6 days. Both 2 X 10(7) and 1 X 10(7) tumor cells injected intraperitoneally will constantly produce tumor nodules in non-MHV-infected nude mice with similar survival. This experimental model has proven useful for in vivo study to assess the immunoreactivity of melanoma patient cells reactive against target tumor cells.

High-molecular-weight DNAs from 43 human primary tumor tissues were examined by Southern blot hybridization for possible rearrangement and/or amplification of the following protooncogenes: the c-myc, c-erbB-1, N-myc, c-mos and c-fos genes. In an adenosquamous cell carcinoma of the stomach, the c-myc and c-erbB-1 genes were found to be simultaneously amplified 5- and 30-fold, respectively. Cooperative expression of the amplified c-myc and c-erbB-1 genes might be involved in the genesis or progression of the gastric cancer.

A total of twenty six peritoneovenous shuntings were performed in our department between 1978 and 1984, on twelve cirrhotic and ten cancerous patients with intractable ascites, using Pudenz, LeVeen or Denver type shunting tubes. Reduced ascites was noted postoperatively with statistical significance in both the cirrhotic group (p less than 0.05) and the cancerous group (p less than 0.01), of whom eight cirrhotic and seven cancerous patients exhibited an abdominal girth reduced by 9.7 cm (a 10.9 per cent reduction) in two weeks. A larger urinary output was noted in 13 patients, with a mean increase of 587 ml/day compared with the pre-shunting output. These patients lost a mean weight of 6.13 kg in two weeks, while the nonreduced ascites group gained weight. The post-shunting serum albumin level was higher in the reduced ascites group. The mean functioning periods of the shunts were 5.1 and 2 months, respectively for the cirrhotic and cancerous patients whose shunts were patent. The patients' outcome was shown to depend on the disease itself, with the exception of one patient who died of disseminated intravascular coagulopathy following shunting. In comparing several types of shunting systems, the Denver-type was proven as being the most unlikely to plug or malfunction because of its simpler structure.