Locoregional immunotherapy of malignant effusion from colorectal cancer using the streptococcal preparation OK-432 plus interleukin-2: induction of autologous tumor-reactive CD4+ Th1 killer lymphocytes.

In total, 16 patients with cytologically proven malignant effusion from colorectal cancer were treated by locoregional administration of the streptococcal preparation OK-432 alone or OK-432 plus the T-cell growth factor interleukin (IL)-2, and the action mechanism of the treatment was studied. A positive clinical response, showing a cytologic disappearance of cancer cells and decrease of effusion, was observed in nine of 11 (82%) patients treated with OK-432 alone and in all five patients treated with OK-432 plus IL-2. Flow cytometric analysis revealed that OK-432 plus IL-2 locally induced acute inflammation-like responses, including serial cellular infiltrations of granulocyte migration within a matter of hours, and activation of macrophages and T lymphocyte involvement within the following days, and that a predominant expansion of CD3+CD4+ lymphocytes (CD: cluster of differentiation) was induced by in vitro stimulation with IL-2 of locoregional cells after the OK-432 administration (OK/IL-2AK cells). The OK/IL-2AK cells produced tumour necrosis factor-alpha and interferon-gamma, but these cells did not produce IL-4 and IL-6. The OK/IL-2AK cells expressed potent killing activity against autologous tumour cells. This activity was abrogated by treatment of the lymphocytes with anti-CD3, -CD4, -TCRalphabeta antibody, and by the treatment of target cells with anti-human leukocyte antigen (HLA)-DR antibody. The OK/IL-2AK cells expressed Fas-L gene, and flow cytometric analysis demonstrated HLA-DR expression in approximately 75% of CEA+ or cytokeratin+ effusion cells. TCRVbeta gene analysis of the OK/IL-2AK cells showed an oligoclonal usage of TCRbeta20, which was also involved in the cytotoxic mechanism of the OK/IL-2AK cells. Single-strand conformational polymorphism analysis demonstrated the clonotypes for the TCRVbeta20 gene, and the CDR3s of the gene were sequenced. The clonotypic PCR using the TCRVbeta20-CDR3 sequences could detect the CDR3-identical TCRs in effusion lymphocytes from the other patients. Taken together, it is suggested that locoregional administration of OK-432 plus IL-2 is highly effective for the management of malignant effusion from colorectal cancer. OK-432 plus IL-2 induces autologous tumour-reactive CD4+ Th1 killer lymphocytes, which recognise tumour antigen(s) presented with HLA class II molecules on effusion tumour cells by means of preferential usage of TCRVbeta20. The clonotypic PCR using the TCRVbeta20-CDR3 sequences may be informative for treating malignant effusion from colorectal cancer using OK-432 plus IL-2.

Kainic acid-induced substantia nigra seizure in rats: behavior, EEG and metabolism.

RATIONALE: In order to clarify the role of substantia nigra pars reticulata (SNr) upon the development of epileptic seizure, kainic acid (KA) was injected into a unilateral SNr. MATERIALS AND METHODS: Wistar rats weighing 250-350 g were used. A stainless-steel cannula and depth electrode were inserted stereotaxically into the left substantia nigra pars reticulata (SNr). At 7 days after surgery, 1.0 microg of KA was injected into the left SNr. Experiment 1: In eight rats, behavior and electroencephalograms (EEG) were continuously recorded for about 30 h, and intermittently monitored following 1 month. Experiment 2: Two hours after KA injection into SNr, rats demonstrated status epilepticus. Then, 100 microCi/kg of [(14)C]2-deoxyglucose (2-DG) was intravenously injected in seven rats, and the rats were processed for autoradiographic study. RESULTS: Changes in behavior and EEG: On EEG, a secondary generalized seizure status was observed at about 70 min after KA injection. In video, limbic seizure manifestations such as salivation were observed as a initial symptom and followed by rolling and generalized tonic seizures. [(14)C]deoxyglucose autoradiographic study demonstrated increased local cerebral glucose metabolism in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular formation. SUMMARY: The result suggested that the substantia nigra played an important role in the secondary generalization in the substantia nigra seizure model due to the decreased function of the GABAergic projection system induced by an excessive epileptic excitation of SNr.

[A case of inoperable advanced gall bladder cancer responding to intra-arterial infusion of 5-fluorouracil (5-FU) and leucovorin (LV)].

The prognosis in cases of inoperable advanced gall bladder cancer is poor. We report here a case of inoperable advanced gall bladder cancer that responded to treatment with continuous intra-arterial infusion of 5-FU and bolus injection of LV for biochemical modulation. The patient was an 81-year-old woman, who visited a nearby clinic with the chief complaints of general fatigue and right lateral abdominal pain. A mass lesion which occupied from the dorsal surface of the liver to the pancreatic head was found by ultrasonography, and she was referred to our hospital for further diagnosis and therapy. The diagnosis was advanced gall bladder cancer of Stage IVa (S2, N3, P0, H0, Hinf1, Dinf1). For the selective arterial infusion of anticancer drugs, the patient underwent intra-arterial cannulation into the common hepatic artery, with a connecting subcutaneous port for arterial infusion therapy. The treatment schedule for 5-FU and LV therapy consisted of continuous infusion of 5-FU of 333 mg/m2 for 72 hr and bolus injection of LV of 20 mg/m2 3 times at 24 hr intervals. This treatment was repeated every 2 weeks. No side effects were observed after the first administration during hospitalization, so the treatment was continued up to 17 times on an outpatient basis. A tumor response was seen in the primary lesion, No. 8 and No. 16 lymph node metastases. A partial response was observed for 13 months and the overall survival was 15 months. These findings may imply that treatment with intra-arterial infusion of 5-FU and LV can be an effective chemotherapy for prolongation of survival in patients with inoperable advanced gall bladder cancer.

Basic science and epilepsy: experimental epilepsy surgery.

Epilepsy surgery, as is employed for the management of intractable seizures, was performed in animals harboring a seizure focus induced by a local application of kainic acid (KA). Amygdalo-hippocampectomy failed to stop spontaneous seizures in the contralateral hippocampus. Callosotomy inhibited seizure propagation to the contralateral sensori-motor cortex. However, epileptic activity ipsilateral to the focus, including subcortical structures, persisted even after the callosotomy. Multiple subpial transection (MST) around the epileptic cortical focus suppressed the seizure activity of the cortex. However, seizure propagations in subcortical structures remained, even after MST. Nefiracetam (a new nootropic agent) was tested in these models, and its promising effect on the intractable extratemporal epilepsy is reported.

[Locoregional immunotherapy of malignant effusions for colorectal cancer using OK-432 and its acting mechanisms--possibility of molecular diagnosis using TCRCDR 3 sequence].

Locoregional administration using OK-432 was evaluated in treating malignant effusion. Positive clinical responses were seen in 19 (52%) of 36 gastric cancer patients, and in 9 (90%) of 10 colon cancer patients (p < 0.05), indicating its clinical benefit in treating malignant effusion of colon cancer. Fever elevation was observed in 43 (93%) patients and local pain occurred with 9 (20%) of 46 administrations. Immunological analysis for responder patients with rectal cancer revealed that OK-432 induced autologous tumor-reactive CD 3+ CD 4+ TCRV beta 20+ killer lymphocytes. The TCR gene analysis permitted us to clone a V beta 20 CDR 3 sequence, by which positive bands were shown in 3 (75%) of 4 responders and negative bands in 3 (100%) of 3 non-responders. It is suggested that cross-antigenicity exists between OK-432 and colon cancer, and that genetic analysis using the TCRCDR 3 sequence makes it possible to predict responder patients to OK-432 immunotherapy.

[Ruptured persistent primitive hypoglossal artery aneurysm: case report].

Persistent primitive hypoglossal artery is less common than persistent primitive trigeminal artery. About one hundred examples of such hypoglossal arteries have been demonstrated by angiography. The origin of persistent primitive hypoglossal artery is the cervical segment of the internal carotid artery, usually at the level of the first to second cervical vertebrae. The artery then enters, with varying degrees of tortuosity, the anterior condyloid (hypoglossal) canal and joints the basilar artery immediately above its lower end. When enlargement of this canal is identified, the presence of a persistent primitive hypoglossal artery should be strongly suspected. The homolateral vertebral artery is frequently hypoplasia. A 66-year-old man was brought to our hospital due to faintness. CT showed thick subarachnoid hemorrhage. Angiography showed that a persistent primitive hypoglossal artery aneurysm was present, but the posterior communicating artery was absent. Right vertebral angiography showed extravasation due to re-rupture of the aneurysm. An operation was performed at day 0 using the left transcondylar approach despite deterioration of SAH grading. Intraoperative re-rupturing occurred and the lower cranial nerves clustered around the aneurysm, so the aneurysm was partially clipped on the dome. The second angiography was carried out at day 10, and there was no vasospasm. Palsy of the lower cranial nerves appeared transiently. A ventricle-peritoneum shunt was required due to normal pressure hydrocephalus, but the patient was discharged with no neurological deficits.

[Usefulness of percutaneous low output microwave tissue coagulation therapy for solitary liver cancer].

The possible use of percutaneous transhepatic low output microwave tissue coagulation therapy (PMCT) using ultra-sonography under local anesthesia for solitary liver cancer was studied. The subjects were 13 patients having primary or metastatic liver cancer with solitary liver tumor less than 3 cm in diameter, including 7 hepatocellular carcinomas and 6 metastatic liver cancers. PMCT was performed continuously 3 times at an output of 30 watts for 30 seconds at a time. Tumors less than 3 cm in diameter were completely coagulated by irradiation from 2 to 6 times, judging by enhanced CT. No tumor recurrence was recognized in the coagulation area. However, in two cases of metastasis from pancreatic carcinoma, multiple metastases were found at another site in the liver by 2 months after PMCT. Thus, the results suggest that PMCT is a useful therapy for small liver tumor as a local control.

[Usefulness of percutaneous microwave tissue coagulation therapy for solitary liver cancer].

The possible use of percutaneous transhepatic microwave tissue coagulation therapy (PMCT) using ultra-sonography under local anesthesia for solitary liver cancer was studied. The subjects were 8 patients having primary or metastatic liver cancer with solitary liver tumor less than 4 cm in size, consisting of 2 hepatocellular carcinomas, and 6 metastatic carcinomas. PMCT was performed continuously 3 times at the output of 30 watts for 30 seconds at a time. Tumors less than 3 cm in size were completely coagulated by irradiation from 2 to 5 times judged by enhanced CT. No recurrence of tumor was recognized in the coagulation area. But in some cases, multiple metastases were found at another site in the liver by 3 months after PMCT. Thus, the results suggest that PMCT is a useful therapy for small liver tumor as a local control.

[A case of advanced hepatocellular carcinoma with portal invasion effectively treated by continuous intra-arterial chemotherapy with 5-fluorouracil].

A 79-year-old male diagnosed as advanced hepatocellular carcinoma with portal invasion was treated by continuous intra-arterial chemotherapy of 5-FU, which was continuously administered for 72 hours at a dose of 333 mg/mm/day every 2 weeks and repeated 12 times. Total dose of 5-FU was 14.4 g. Toxicity of this therapy was not recognized. Levels of AFP were reduced from 4385.6 ng/ml to 11.9 for 6 months. No tumor was recognized on CT scan at 6 months after starting this therapy, after 15 months of this therapy, the patient is alive and disease-free. Given the above results, continuous intra-arterial administration of this 5-FU therapy may be effective for patients with hepatocellular carcinoma.

Prevention of CSF Leakage by Staged Operation for Clival Metastatic Tumor.

The transoral approach is a direct route to the clivus. However, application of this approach is infrequent because of the risk of cerebrospinal fluid (CSF) fistula and subsequent meningitis. We report a case of clival metastatic tumor treated by staged operation without CSF leakage. A 39-year-old man was found to have a tumor in clivus extending to the intradural space. Two-staged resection through the lateral suboccipital and transoral approach was performed and the dural defect was replaced by a fascia in the first operation. CSF leakage was prevented by this procedure. The patient received radiotherapy postoperatively.

[Usefulness of subcutaneous implantable port with new one-way valve].

Because of the development of the subcutaneous implantable port, intra-arterial infusion chemotherapy for malignant tumor has been performed in safety and it is now possible to choose a more effective method of drug administration. Many problems remain, however. In this study, we investigated the performance of subcutaneous implantable port with a new one-way valve with safety, septum intensity and a back stream of blood. This new port was very safe, with good septum intensity and water backflow. In the performance test with a back stream of water into the catheter, water did not back up into the catheter connecting with the port with the one-way valve, but did so without the one-way valve after releasing a load of 2,000 g from the septum. This suggests that the catheter obstruction is decreased by adding a one-way valve to the port.

Transfection of human cytochrome P-450 reductase cDNA and its effect on the sensitivity to toxins.

NADPH-cytochrome P-450 reductase (CYPR; EC 1.6.2.4) is an essential enzyme for the catalytic function of cytochromes and is also regarded as being implicated in drug activation. To examine whether CYPR is directly involved in the drug metabolism, a cDNA encoding human CYPR was stably transfected into Chinese hamster ovary cells. Three clonal cell lines were identified which expressed increased levels of CYPR activity (9.3- to 11.2-fold). Western blot analysis indicated that CYPR-transfectant cells contained markedly elevated levels of CYPR protein, while wild-type Chinese hamster ovary cells contained low levels. They showed 1.8- to 3.3-fold higher sensitivity to Adriamycin, 2.1- to 3.0-fold higher sensitivity to mitomycin C, and 2.9- to 4.3-fold higher sensitivity to paraquat than wild-type cells. We also studied other common-use anticancer agents: vinblastine, vincristine, VP-16, and cisplatin, but there were no differences observed in the sensitivity. This report provides the first evidence that transfection of human CYPR into mammalian cells is concerned in the sensitivity to some drugs.

[A case of Jefferson fracture treated with the Sof'wire cable system method of fixation].

Jefferson fracture is a very rare disease which occurs in only 2-13% of all cervical spinal fracture cases and in only 1.3% of total spinal fracture cases. A combination of an atlas-axis fracture occurs relatively frequently and with a higher incidence of neurological morbidity than isolated fractures. However, a Jefferson fracture, which is an isolated C-1 fracture, occurs very rarely. A 58-year-old woman was involved in a traffic accident and admitted to our hospital. She had a large scalp laceration in the parietal region and complained of nuchal pain. Neurological examination revealed nothing abnormal. A cervical x-p (lateral view) revealed no abnormal findings, but an open-mouth view revealed an 8 mm displacement (in total) in the lateral mass of the atlas. A cervical CT revealed a Jefferson fracture. Crutchfield traction was performed for 9 days followed by external immobilization with a halo-vest to allow the patient to be ambulatory quickly. A posterior occipitocervical fusion was performed with an iliac bone autograft using the Sof'wire Cable system for late cervical stability and reducing the period of rigid external immobilization. The postoperative state was uneventful. The halo-vest was removed 10 weeks after surgery. An x-p obtained 3 months postoperatively showed good stability of the cervical spine. The Sof'wire Cable system proved to be very useful.

[T cell receptor V beta repertoire of locoregional lymphocytes in malignant effusions].

We analyzed the T cell receptor (TCR) V beta repertoire of lymphocytes obtained from patients with malignant effusion treated by locoregional immunotherapy. Polymerase chain reaction using a panel of V beta subfamily specific oligonucleotide primers(V beta 1-20) was employed after reverse transcription of mRNA isolated from the locoregional cells. No major oligoclonality of TCR repertoire was observed in effusion lymphocytes before the treatment. The expression level of V beta 13.1 repertoire was significantly higher in effusion lymphocytes than in peripheral blood lymphocytes before treatment. V beta 20 gene expression increased significantly after locoregional administration of OK-432. It is suggested that TCR V beta 13.1 may be responsible for effusion lymphocytes and TCR V beta 20 for OK-432-related antigenic stimulus.

Locoregional immunotherapy of malignant ascites by intraperitoneal administration of OK-432 plus IL-2 in gastric cancer patients.

The clinical efficacy of intraperitoneal administration of OK-432 plus interleukin-2 (IL-2) for treating malignant ascites was evaluated in gastric cancer patients. Ten KE of OK-432 and 200,000 Jurkat units of IL-2 were intraperitoneally administered in tandem in the order given on alternate days at paracentesis. Of the 22 evaluable patients, 18 (81%) developed complete or partial responses, showing a cytologic disappearance of cancer cells and decrease of ascites. More than 50% of the patients obtained positive responses within 2 weeks after the initial administration of the drugs. Improvements of performance status and clinical symptoms such as abdominal fullness, followed by restoration of oral food intake and prolongation of survival time were observed in responders treated with OK-432 plus IL-2. Flow cytometric analysis demonstrated a predominant increase of the CD3+CD4+ cells, especially of the CD4+CD45RA- subset in the peritoneal cavity of the responders. Cytotoxicity assay after negative selection of the CD4+ cells with the antibody and complement revealed that the CD4+ subset possessed cytotoxic activity against autologous tumor cells. The results suggest that intraperitoneal administration of OK-432 plus IL-2 may not be only a practical but also an effective protocol for treating malignant ascites in gastric cancer patients.

[Skin reaction to OK-432 and its dosage for locoregional administration].

Establishment of optimal dosage of OK-432, a streptococcal preparation, was studied based on its skin test was studied. Locoregional immunotherapy using OK-432 was conducted for patients with malignant fluids. More OK-432 was administered to patients having a weaker skin reaction to OK-432 and less to those having a stronger one, corresponding to their redness diameter of skin reaction. There was a marked difference in OK-432-skin test among patients. Some patients with malignant fluids having small redness responded to the treatment after a large amount of OK-432, and others were well controlled by a smaller dosage of OK-432. It is suggested that OK-432-skin test may provide the optimal dosage for local treatment of malignant fluids.

[Intra-arterial infusion chemotherapy of cisplatin (CDDP)-lipiodol suspension using implantable injection port for unresectable liver cancer patients].

Intra-arterial CDDP-Lipiodol infusion chemotherapy using an implantable port was effective in 10 unresectable liver cancer patients, including 7 hepatocellular and 3 metastatic cases. CDDP-Lipiodol suspension (10 mg of CDDP/1 ml of Lipiodol) was administered at the dose of 25 mg/m2 of CDDP biweekly from 2 to 9 times. The clinical responses were defined as 4 PR (40%), 5 NC (50%), including 3 MR, and 1 PD (10%). The efficacy rate was 40%. The level of AFP and CEA was reduced in all PR and NC cases except one. Side effects were nausea (70%), low-grade pyrexia (50%), abdominal pain (30%), and liver dysfunction (20%), but they were tolerable and transient.

Catalytic properties of rabbit kidney fatty acid omega-hydroxylase cytochrome P-450ka2 (CYP4A7).

We have examined in detail the substrate specificity of a rabbit kidney fatty acid omega-hydroxylase, designated cytochrome P-450ka2 (CYP4A7). The hydroxylation products were identified as omega- and (omega - 1)-hydroxy fatty acids mainly using gas chromatography-electron impact mass spectrometry. [1] Straight-chain saturated fatty acids ranging from 10 to 19 carbons were effectively hydroxylated at the omega- and (omega - 1)-position. The ratios of omega- to (omega - 1)-hydroxylation activity decreased with increasing the carbon chain length of fatty acids. [2] Both isomyristate and anteisomyristate, and isopalmitate were hydroxylated several fold more rapidly than myristate and palmitate, respectively, with iso-branched chain fatty acids being hydroxylated at the omega-position solely. [3] Both palmitoleate and palmitoelaidate, and both oleate and elaidate were hydroxylated much more rapidly than palmitate and stearate, respectively. [4] Linoleate, gamma-linolenate, and arachidonate were also excellent substrates for this enzyme. [5] Prostaglandin (PG) A1 and PGA2 were efficiently hydroxylated at the omega-position solely, with PGE1 and PGE2 being much less active. [6] Arachidonic acid not only showed a Km value significantly lower than those for lauric acid, gamma-linolenic acid and PGA1, but also it is a potent competitor for lauric acid and PGA1, showing a very high affinity for the enzyme. It is possible that arachidonic acid is the physiological substrate for kidney P-450ka2.

The role of lymphocyte surface binding sites for wheat germ agglutinin in the negative regulation of cancer patients.

The role of lymphocyte surface binding sites for wheat germ agglutinin (WGA) in the negative regulation of cancer patients was investigated. The number of WGA binding sites on the surface of each lymphocyte ranged from 10(7) to 10(8). Fluorescein isothiocyanate (FITC)-conjugated WGA, bound to the majority of peripheral blood lymphocytes (PBL) with two peaks of fluorescent intensity was expressed either dimly or brightly. The increase in lymphocytes brightly expressing WGA fluorescent intensity (WGA bright lymphocytes) significantly correlated with the number of WGA binding sites. The suppression of lymphocyte proliferation mediated by the purified soluble suppressor factor (SSF) significantly correlated with an increase in the WGA bright lymphocyte population (P < 0.05). A significantly greater number of WGA bright lymphocytes in PBL was found in patients with esophageal, gastric, breast, or colon cancer, than in those with benign diseases or in healthy controls. Furthermore, an increase in WGA bright lymphocytes was found in subsets expressing the antigens CD8 dimly or CD16. Thus, it is suggested that the number of WGA binding sites may increase mainly on the surface of effector cells such as NK cells and CD8-positive killer T cells in cancer patients, triggering the negative regulation mediated by SSF.