[Local injection of BRM-activated killer cells into an abdominal wall tumor].

Based on the concept of living with cancer, wherein the goal is to help patients with highly advanced solid cancers maintain a high quality of life(QOL)without adverse events and drug resistance, we developed a new immunocyte therapy based on BRM-activated killer(BAK)cells, which are primarily CD56 positive lymphocytes. In a previous report, we documented the disappearance of liver metastases, as assessed by positron emission tomography-computed tomography(PET-CT), in patients with metastatic liver cancers into which BAK immunocytes had been administered via injection into the hepatic artery. Herein, upon the patient's request, we locally injected BAK lymphocytes into an abdominal tumor. In BAK therapy, 20 mL of peripheral blood are collected from a patient. Lymphocytes from this blood sample are subsequently activated and multiplied with immobilized anti-CD3 antibodies and IL-2 and are cultured for 2 weeks with E(bina)and serum-free ALys media to yield approximately 10 billion autologous lymphocytes. On the final day of incubation, the lymphocytes are treated with 1,000 units/mL of interferon(IFN)-a for 15 minutes to enhance their therapeutic killing effects. During the second week, approximately 10 billion isolated autologous lymphocytes are suspended in 200 mL of Ringer's solution and are then drip-infused into the patient over a period of 1 hour. We injected approximately 10 billion BAK lymphocytes suspended in 50 mL of Ringer's solution into a 2-cm abdominal tumor in a single 60-year-old woman under ultrasonography guidance. This procedure was repeated every 3 weeks. After the third administration, we collected a biopsy specimen and examined it using PAS staining and microscopy. The 3 separate local injections of approximately 10 billion activated autologous lymphocytes each, primarily CD56 positive cells, into the tumor led to tumor fragmentation, leaving approximately 10 lymphocytes surrounding each cancer cell. These results suggest that BAK therapy is efficacious and show that locally administered BAK lymphocytes can reach cancer tissues and effectively kill cancer cells.

[Hepatic intra-arterial infusion of biological response modifier (BRM)-activated killer immune lymphocytes to treat liver cancer].

To enable "life with cancer" while maintaining quality of life(QOL) and freedom from adverse effects, we have devised biological response modifier (BRM)-activated killer (BAK) therapy, a new immune cell therapy using CD56-positive lymphocytes designed to treat advanced solid cancers. This treatment is yet to be applied to liver cancer, because BAK cells administered by intravenous infusion are unlikely to reach lesions via the complex vascular and lymphatic systems. Here, we report a case in which the patient, a surgeon, requested that we attempt to deliver BAK cells to cancer lesions in the liver via hepatic intra-arterial infusion. In 2005, with the patient's consent, we injected autologous lymphocytes intra-arterially into the liver using a catheter and confirmed the absence of adverse effects. In December 2008, we began injecting BAK cells intra-arterially into the liver of the patient, a male physician aged 52 years at the time, after identifying liver metastases following surgery for rectal cancer. We injected 10 billion cells intra-arterially into the liver via a catheter once a month for 6 months, and then undertook conventional BAK therapy by monthly intravenous infusion. Based on images obtained by positron emission tomography-computed tomography, BAK therapy led to complete remission and disappearance of the liver metastases. This case highlights the effectiveness of hepatic intra-arterial infusion of BAK cells in cases of liver cancer.

[Hepatic intra-arterial infusion of BAK immune cells to treat metastatic liver cancer].

Based on the "living with cancer" concept while maintaining a favorable QOL and avoiding side effects and drug resistance, we have developed a new immune cell treatment called BAK (BRM activated killer) therapy, primarily using CD56+ cells for a case of advanced progressive solid cancer. In the present case, we administered BAK cells by hepatic intra-arterial infusion to a patient who happened to be a surgeon and wished to undergo this therapy. The patient was a 52-year- old male surgeon who underwent surgery for rectal cancer in April 2007. Heavy particle radiotherapy was administered when liver metastases were identified in July 2008. Starting in December 2008, 10 billion BAK cells were administered each month by hepatic intra-arterial infusion via a catheter on a total of six different occasions. The 10 billion autologous lymphocytes were suspended in 200 mL of Ringer's solution and returned to the patient by hepatic intra-arterial infusion over a period of one hour. Interactions between the activated lymphocytes and liver cancer cells increased levels of serum α1AG, an inflammation marker, but these levels normalized following the sixth and final administration. Conventional drip-infusion BAK therapy was administered thereafter. Diagnostic imaging, including PET-CT and PET, confirmed a complete disappearance of liver metastases. This case suggests the effectiveness of hepatic intra-arterial infusion BAK cell therapy in treating liver cancer.

The bovine lactophorin C-terminal fragment and PAS6/7 were both potent in the inhibition of human rotavirus replication in cultured epithelial cells and the prevention of experimental gastroenteritis.

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M(r) glycoprotein fraction (F1) from cow's milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 degrees C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

Cell transfer regimens in patients with highly advanced surgically unresectable non-small cell lung cancer: significantly improved overall survival in patients with lower levels of serum immunosuppressive acidic protein.

Sixty-one non-small cell lung cancer (NSCLC) patients with stage II and III/IV were enrolled and 49 completed immunotherapy. Patients were grouped based on immunosuppressive acidic protein (IAP). All patients received monthly intravenous infusions containing 1 x 10(10) (mean cell number per patient) ex vivo expanded and IFN-alpha-treated peripheral blood mononuclear cells. No patients had grade 2 or greater adverse events. The patients with < or = 580microg/ml of serum IAP levels (n=33) had significantly longer recurrence-free survival than those with > 580microg/ml of serum IAP levels (n=16). Patients with lower IAP levels are still under immunotherapeutic control after 27 months free of recurrence. The IAP levels may be a prognostic marker for treatment efficacy in NSCLC. This immunotherapeutic regimen was feasible and well tolerated in patients with advanced NSCLC in terms of prolongation of survival.

[Non-small cell lung cancer--immunocell BAK (BRM activated killer) therapy].

We enrolled in this study 14 immunosuppressed non-small cell lung cancer (NSCLC) outpatients whose IAP level in serum were over 580 microg/ml and 26 immunoreactive outpatients whose IAP level in serum were under 580 microg/ml. After giving informed consent, patients were treated with BAK therapy on an outpatient basis. Treated with BAK therapy, the mean survival of immunosuppressed patients was 5.2 months, on the other hand, one of immunoreactive patients was 26.3 months. The difference in survival between 2 groups was significant (p<0.01). A stage IV NSCLC patient whose serum IAP is under 580 microg/ml is good indication for BAK therapy. The favorable clinical response in lung cancer patients to BAK treatment may be due to the fact that lungs are the first exposed to BAK cells via the blood stream. BAK therapy has a life prolonging effect with no apparent adverse effects for advanced NSCLC patients.

[Antitumor effects of intratumoral injection of Basidiomycetes preparations].

The antitumor effects of Basidiomycetes preparations in an experimental mouse model, the "double grafted tumor system" were analyzed. Some BRMs prevented metastases by utilizing the anti-tumor immunological cascade reactions, which activates macrophages in the body. The following Basidiomycetes preparations were analyzed: PSK was a hot water extract of cultured mycelia from Coliolus versicolor and a protein bound beta-glucan. Matsumax was extracted from mycelia of Tricholoma matsutake and was a protein bound (38%) a-glucan. The Agaricus preparation was extracted from fruit bodies of Agaricus blazei and a protein-bound (17%) a-glucan, beta-glucan. Himematsutake preparation was extracted from fruit body of Agaricus blazei (Himematsutake) and a protein bound (5%) glucan. Lentinan was purified from fruit bodies of Lentinus edodes and is a purified beta-glucan. PSK cured both primary and metastatic tumors in the double grafted tumor system. Lentinan inhibited the growth of neither primary nor metastatic tumors. Matsumax and Agaricus preparation cured primary tumor and inhibited the growth of metastatic tumor. Himematsutake preparation inhibited the growth of primary tumor. Immunosuppresive acidic protein (IAP) is produced by activated mactrophates. The PSK, Matsumax, Agaricus preparation and Himematsutake preparation induced IAP but Lentinan did not.

[Life-prolonging effect of immunocell BAK (BRM activated killer) therapy--evidence based integrative medicine].

We devised an innovative type of immunocell therapy called biological response modifier (BRM)-activated killer (BAK) therapy, which utilizes most of non-MHC (major histocompatibility complex)-restricted lymphocytes, CD56-positive cells including gammadelta T cells and NK cells. CD56-positive cells are neuro-immune-endocrine (NIE) multifunctional, integrated cells. We enrolled 30 immunosuppressed patients whose immunosuppressive acidic protein (IAP) levels in serum were over 580 microg/ml, and 63 immunoreactive solid cancer outpatients whose IAP level in serum were under 580 microg/ml. Treated with BAK therapy, the mean survival time of immunosuppressed patients was 5.0 months. On the other hand, survival time of immunoreactive advanced postoperative patients (stage IV) and inoperable lung cancer patients (stage IIIb) was 27.1 months. BAK therapy has a life-prolonging effect without any adverse effects and maintains satisfactory quality of life (QOL) for advanced solid cancer patients. Based on this evidence, we propose what can be called Integrative medicine which is neither Western nor Chinese medicine.

[Activation of antitumor immunity by intratumor injection of biological preparations].

The antitumor effects of biological response modifiers (BRMs) in an experimental mouse model using a double grafted tumor system were analyzed. Some BRMs prevented metastases by utilizing the anti-tumor immunological cascade reactions, which activate macrophages in the body. The following BRMs were analyzed: PSK was a hot water extract of cultured mycelia from Coliolus versicolor and a protein bound beta-glucan. Lentinan was purified from fruit bodies of Lentinus erodes and is a beta-glucan. The agaricus preparation was extracted from fruit bodies of Agaricus blazei and a protein-bound alpha-, beta-glucan. The M2 fraction was extracted from mycelia of Tricholoma matsutake and was a protein bound alpha-glucan. M1 fraction was purified from mycelia of T. matsutake and was an alpha-glucan. PSK cured both primary and metastatic tumors in the double grafted tumor system. Lentinan did not inhibit the growth of either primary or metastatic tumors. Agaricus preparation cured a primary tumor and inhibited the growth of a metastatic tumor. The M2 fraction prepared from Matsutake inhibited the growth of both primary and metastatic tumors. The M1 fraction did not inhibit either primary or metastatic tumors. Immunosuppressive acidic protein (IAP) is produced by activated macrophages. The PSK, Agaricus preparation and M2 fraction of the Matsutake preparation induced IAP but the lentinan and M1 fraction did not.

Life-prolonging effect of immunocell BAK (BRM-activated killer) therapy for advanced solid cancer patients: prognostic significance of serum immunosuppressive acidic protein levels.

We devised an innovative type of immunocell therapy called BRM (biological response modifier)-activated killer (BAK) therapy, which utilizes most of non-MHC (major histocompatibility complex) restricted lymphocytes, CD56+ cells including gammadelta T cells and NK cells. Peripheral blood lymphocytes were selected by immobilizing them with anti-CD3 monoclonal antibody, cultured for 2 weeks with serum-free medium containing IL-2, and then were reactivated by 1,000 U/ml of IFN-alpha for 15 min. The patients were infused with about 6x10(9) BAK cells by intravenous drip infusion at 1-month intervals. All advanced solid cancer patients who had received chemotherapy but for whom it was not effective or have refused chemotherapy were included in the present study. A good marker of impairment of host immune response by chemotherapy is an immunosuppressive acidic protein (IAP) level in serum above 580 microg/ml; survival rates were compared with the high (> 580 microg/ml) and the low (< or = 580 microg/ml) serum IAP groups. We enrolled in this study 23 immunosuppressed patients whose IAP levels in serum were over 580 microg/ml, and 42 immunoreactive solid cancer outpatients whose IAP level in serum were under 580 degreesg/ml and whose performance statuses were over 80% on the Karnofsky scale. After giving informed consent, patients were treated with BAK therapy on an outpatient basis at our hospital. The ethical review board of the Miyagi Cancer Center approved this pilot study. Treated with BAK therapy, the mean survival of immunosuppressed patients was 4.6 months. On the other hand, survival for one of immunoreactive advanced postoperative patients (stage IV) and inoperable lung cancer patients (stage IIIb) was 24.7 months. The difference in survival between the 2 groups was significant (P < 0.01). This shows that BAK therapy is not indicated for an advanced cancer patient whose serum IAP is over 580 microg/ml, perhaps due to extensive chemotherapy. Overall response to BAK therapy was complete response (CR) in 5 cases, partial response (PR) in 1 case, and prolonged no change (NC) in 26 cases, with an effectiveness rate at 76.2% in 42 advanced stage IIIb and IV cancer patients. BAK therapy has a life-prolonging effect without any adverse effects and maintains satisfactory quality of life (QOL) for advanced cancer patients.

Occult lymphoma cells prevalent in autologous marrow from non-Hodgkin's diffuse lymphoma.

The most effective treatment for recurrent non-Hodgkin's lymphoma (NHL) appears to be a high-dose cytotoxic chemotherapy (HDC) followed by autologous bone marrow transplantation (ABMT). However, it has been suggested that the presence of occult lymphoma cells in harvested marrow may be responsible for a significant fraction of treatment failures after HDC/ABMT. The present study examined randomly accrued NHL patients, independent of their cytogenic grades, for the presence of cells bearing bcl-2/immunoglobulin heavy chain (IgH) gene rearrangements in lymph node (LN) biopsies and the bone marrow by polymerase chain reaction (PCR) and Southern blot hybridization combined with a classical culturing technique. Among 41 NHL patients examined, bcl-2/IgH translocations were evident in LN biopsies and marrow from each of 10 follicular lymphoma patients, but not in any samples from 31 newly diagnosed diffuse lymphoma patients. Marrow aspirates from several patients that were cultured using a one-week "triggering culture" followed by an extended period of conventional culture resulted in emergence of a monoclonal, IgH-rearranged, bcl-2-normal lymphoid cell population. Such outgrowth was specifically seen in cultures of diffuse lymphoma marrow (7 of 28 evaluable patients). Southern analysis for IgH rearrangement within LN biopsies and of cells cultured from marrow of individual diffuse lymphoma patients produced identical patterns, suggesting that the occult lymphoma cells present in harvested marrow were derived from the predominant lymphoma cell population represented within involved lymph nodes. The culture of histologically occult lymphoma from diagnostic marrow and analysis of the derived cells by Southern blot hybridization can be used to detect potentially aggressive lymphoma cells within harvested marrow, despite their lack of bcl-2 gene rearrangement.

Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy.

Interleukin (IL)-2 can induce large numbers of lymphokine-activated killer cells in peripheral blood lymphocytes (PBL), but IL-2 alone cannot induce proliferation of a large number of canine (c) PBL. We used the solid phase anti-CD3 antibody and soluble recombinant (r) IL-2 in order to establish a large scale culture method for cPBL. The number of lymphocytes seeded (3 x 10 (7)) increased to 1 x 10(9) after incubation for 10 days. The phenotype of cultured cPBL cells (after 2 weeks) showed a CD4(+) or CD8(+) predominant cell population. The cultured cell solutions were administered with physiological saline intravenously to each dog. After transfusion of the cultured cells, the cPBL counts, especially the number of CD4(+), CD8(+) and CD4(-)CD8 (-)(DN) cells increased significantly in the recipient dogs. Natural killer (NK) cells, gammadeltaT cells and B cells were considered to be present in the DN cell population. The NK cells and gammadeltaT cells showed no adverse reaction to the transfusion of the activated cPBL. Therefore, it is necessary to recognize the B cells present in the DN cell population by detecting CD21(+) cells. In conclusion, the bulk culture system of cPBL with rIL-2 and solid phase anti-CD3 antibody may be useful for the development of novel immunotherapy in dogs.

Orally administered beta-1,6-D-polyglucose extracted from Agaricus blazei results in tumor regression in tumor-bearing mice.

There is an increasing demand from both patients and practicing oncologists for orally formulated chemotherapy. The present study focused on the oral formulation for natural products that may be effectively used in oncologic treatment regimens. Tumor-bearing mice treated with intratumoral administration of aqueous ammonium oxalate-soluble and ethanol-insoluble derivatives of Agaricus blazei showed marked tumor regression at doses ranging from 0.1 to 2.5 mg (p < 0.05 vs. saline control; n = 7). However, oral administration of this same fraction, either prior to, simultaneously with, or after, tumor cell inoculation did not result in tumor regression (p > 0.05 vs. control). When this fraction was treated with hydrochloric acid (acid-treated fraction; ATF), intratumoral administration resulted in a marked regression of tumor growth comparable to that of the acid-untreated fraction. More importantly, parenteral administration of ATF resulted in a significantly greater regression of tumor growth than that produced by the untreated fraction (p < 0.05 vs. untreated; n = 7). When a total of 4.5 mg of ATF was given orally at varying schedules prior to, simultaneously with, or after, tumor inoculation, a significant regression was seen using a schedule starting 4 days prior to inoculation (p < 0.05 vs. all other treatments; n = 7). NMR and molecular analyses showed that the ATF fraction had a molecular weight of approximately 10 kDa and consisted mainly of only (1,6)-beta- D-polyglucose. These results suggest that the oral administration of simple acid-treated ATF results in a remarkable tumor regression. Thus, simple acid hydrolysis of natural products may not only bring measurable benefits in oncological practice, but may also be a useful general formulation for natural products for oral chemotherapy.

Protective efficacy of a sulfated sialyl lipid (NMSO3) against human rotavirus-induced diarrhea in a mouse model.

Antiviral activity of sulfated sialyl lipid (NMSO3) against human rotavirus (RV) was examined in vitro and in vivo. NMSO3 inhibited the replication of four major serotypes (G1 to G4) of human rotavirus with a low 50% effective concentration of 1 to 5 microg/ml and 50% cytotoxic concentration of 153 microg/ml when determined by plaque assays with MA104 cells. Exposure of NMSO3 to HCl (pH 2.0) for 30 min exhibited no loss of anti-RV activity. Time-of-addition experiments revealed that NMSO3 inhibited the adsorption of four serotypes of RV to MA104 cells. Furthermore, an assay of virus binding with radiolabeled RVs revealed that NMSO3 inhibited the binding of virus to MA104 cells, suggesting that NMSO3 may bind to VP4 and/or VP7. Prophylactic oral administration of NMSO3 (10 microg three times per day, 4 days) to five suckling mice starting 30 min before inoculation of MO strain (3 x 10(6) PFU/mouse) prevented the development of diarrhea. Four of five mice showed no stool or brown formed stool, and only one mouse showed brown soft stool, while water treatment caused watery diarrhea in all five mice. The mean titer of antibody to RV in mice which received NMSO3 at 10 microg three times per day for 4 days was significantly lower than that of untreated, infected mice. NMSO3 is a promising candidate for the prophylactic treatment of human RVs.