Effects of Lactobacillus plantarum Strain OLL2712 Culture Conditions on the Anti-inflammatory Activities for Murine Immune Cells and Obese and Type 2 Diabetic Mice.

Studies on the health-promoting effects of lactic acid bacteria (LAB) are numerous, but few provide examples of the relationship between LAB function and culture conditions. We verified the effect of differences in culture conditions on Lactobacillus plantarum OLL2712 functionality; this strain exhibits anti-inflammatory activity and preventive effects against metabolic disorders. We measured interleukin-10 (IL-10) and IL-12 production in murine immune cells treated with OLL2712 cells prepared under various culture conditions. The results showed that the IL-10-inducing activities of OLL2712 cells on murine immune cells differed dramatically between OLL2712 groups at different culture phases and using different culture medium components, temperatures, and neutralizing pHs. In particular, exponential-phase cells had much more IL-10-inducing activity than stationary-phase cells. We confirmed that the Toll-like receptor 2 (TLR2) stimulation activity of OLL2712 cells depended on culture conditions in conjunction with IL-10-inducing activity. We also demonstrated functional differences by culture phases in vivo; OLL2712 cells at exponential phase had more anti-inflammatory activity and anti-metabolic-disorder effects on obese and diabetic mice than those by their stationary-phase counterparts. These results suggest that culture conditions affect the functionality of anti-inflammatory LAB.IMPORTANCE While previous studies demonstrated that culture conditions affected the immunomodulatory properties of lactic acid bacteria (LAB), few have comprehensively investigated the relationship between culture conditions and LAB functionality. In this study, we demonstrated several culture conditions of Lactobacillus plantarum OLL2712 for higher anti-inflammatory activity. We also showed that culture conditions concretely influenced the health-promoting functions of OLL2712 in vivo, particularly against metabolic disorders. Further, we characterized a novel mechanism by which changing LAB culture conditions affected immunomodulatory properties. Our results suggest that culture condition optimization is important for the production of LAB with anti-inflammatory activity.

Changes in pulmonary histology and exfoliated bronchoalveolar cells induced by in vivo introduction of the tumor necrosis factor-alpha gene.

An inflammatory cytokine, tumor necrosis factor (TNF)-alpha, has been implicated in the pathogenesis of inflammatory lung diseases such as interstitial pneumonia (IP). To clarify the role of the inflammatory cytokine in the pathogenesis of lung inflammation, we introduced a murine TNF-alpha gene into murine lungs by the hemagglutinating virus of Japan (HVJ)-liposome method. Seven days after the TNF-alpha gene introduction resulted in marked cellular infiltration of alveoli, and mild histological change was observed 28 days after the gene introduction. Electron microscopic analysis revealed minimal deposition of collagen fibrils. Analysis of the BAL revealed that the total cell number was markedly increased 3 and 7 days after the gene introduction, and more than 90% of the cells were macrophages. The increase in the cell number was returned to below the normal level 28 days after the gene introduction. During the development of IP, TNF-alpha may regulate pathologic change of the pulmonary interstitium and alveolar cells.

Introduction of plasmid DNA and oligonucleotides into lung epithelial cells by the hemagglutinating virus of Japan (HVJ)-liposome method.

The hemagglutinating virus of Japan (HVJ) fused with liposomes provides a unique transfection vehicle with characteristics of both virus vector and liposome. Here we investigate the efficiency and safety of the HVJ-liposome technique in delivering foreign genes and oligonucleotides into the lung of the Wistar rat. A plasmid vector containing the Escherichia coli beta-galactosidase (beta-gal) gene and the chicken beta-actin promoter was transfected via the trachea using the HVJ-liposome method. Cytochemical staining showed expression of exogenous beta-gal activity in airway epithelial cells, alveolar macrophages, and alveolar type II cells. This activity persisted at least 28 days after administration of the genes. FITC-labeled oligonucleotides also were introduced into the same types of lung cells as those expressing beta-gal. After instillation of HVJ-liposome, anti-HVJ antibodies were detected in the sera of the rats, but even after repeated administration of HVJ-liposome, no marked histopathologic change was observed while exogenous beta-gal expression was detected in pulmonary cells.

[Diffuse alveolar hemorrhage after allogeneic bone marrow transplantation].

A 34-year-old woman with chronic myeloid leukemia underwent allogeneic bone marrow transplantation (BMT) after receiving high-dose chemotherapy and total body irradiation. She experienced progressively dry cough 51 days after BMT, and chest X-ray films showed patchy infiltrations in the lower fields of both lungs on the 66th day after BMT. The symptoms of cough, fever, and hypoxemia worsened. The patchy infiltrations continued to spread and fuse. Diffuse alveolar hemorrhage (DAH) was diagnosed on the basis of high-resolution CT and bronchoalveolar lavage findings. Treatment with high-dose methyl prednisolone pulse therapy, antibiotics, and haptoglobin resolved the patient's DAH symptoms. DAH was thought to be secondary to thrombotic microangiopathy. The majority of patients who experience DAH after BMT eventually die. The remission observed in our case was rare, and illustrated that steroid therapy can be effective for DAH after BMT.

Interleukin-6 secreting phaeochromocytoma associated with clinical markers of inflammation.

Phaeochromocytomas have been shown to produce not only catecholamines but other neuropeptides and hormones, with a variety of clinical manifestations. We report a 70-year-old female patient with phaeochromocytoma exhibiting sustained hypertension, low-grade fever, thrombocytosis, and elevated levels of plasma fibrinogen and C-reactive protein. Serum interleukin (IL)-6 levels were significantly elevated, whereas serum IL-1 alpha and IL-1 beta were not detectable. After surgical removal of the tumour, hypertension and low-grade fever disappeared, and the laboratory finding including serum IL-6 concentrations became normal. Immunohistochemical study of the tumour showed positive staining for IL-6. Culture of the resected tumour revealed the production of large amounts of IL-6. It is suggested that IL-6 secreted by the tumour was responsible for some of the clinical manifestations in this patient.

[Effects of in vivo instillation of genes coding for cytokines on pulmonary fibrosis].

Interstitial pneumonia is characterized by alveolitis that results in interstitial fibrosis. To study the role of humoral factors in the pathogenesis of interstitial fibrosis, we introduced expression vectors into Wistar rats via the trachea, to cause local overexpression of these humoral factors in the lung. Genes for human interleukin (IL)-6 and for the IL-6 receptor caused lymphocytic alveolitis without marked proliferation of fibroblasts. In contrast, overexpression of the genes for human transforming growth factor (TGF)-beta 1 and for human platelet-derived growth factor (PDGF)-B caused only mild cellular infiltration in the alveoli. However, both caused marked proliferation of fibroblasts and deposition of collagen fibrils. Introducing an expression vector that coded for a mutant form of the PDGF beta receptor that lacks its cytoplasmic domain markedly alleviated the pathohistologic changes caused by bleomycin in murine lungs. These findings show that TGF- and PDGF-B may be closely related to fibrosis in the lung, and that artificial regulation of them may be effective for treatment of lung fibrosis.

Lung cancer producing interleukin-6.

A patient with complaints of high fever and left shoulder pain was found to have a large mass in the left upper lobe on chest roentgenogram. Laboratory evaluation revealed marked thrombocytosis, hypoalbuminemia, and increased serum concentrations of CRP, fibrinogen and interleukin-6 (IL-6). A transcutaneous biopsy specimen revealed large cell carcinoma. Tumor production of IL-6 was confirmed by immunohistochemical staining with an anti-human IL-6 monoclonal antibody (MH60).

Antitumor activity of SPM VIII, a derivative of the nucleoside antibiotic spicamycin, against human tumor xenografts.

The antitumor activity of spicamycin analogue SPM VIII against human stomach, breast, lung, colon and esophageal cancers was compared to that of mitomycin C (MMC) in the human tumor-nude mice xenograft model. Comparative studies of SPM VIII given i.v. at 6 mg/kg/day daily for 5 days and MMC given i.v. at 6.7 mg/kg on day 1 revealed that the antitumor spectrum of SPM VIII showed a different pattern from that of MMC and that SPM VIII caused tumor mass reductions in more tumors than did MMC in colon cancers (4/12 versus 1/11). In addition to this study, a comparative study of SPM VIII given i.v. at 12 mg/kg/day 8 times at 3- or 4-day intervals and 5'-deoxy-5-fluorouridine (5'-DFUR) given po at 185 mg/kg/day 5 days per week for 4 weeks showed that SPM VIII had the highest effect on SC-9 human stomach cancer and COL-1 human colon cancer among the 3 compounds, resulting in a significant reduction of tumor mass. Although other pharmacological studies are in progress, these results suggest that SPM VIII might be a novel antitumor compound effective for human cancers including cancer of the digestive organs.

Antitumor activity of a spicamycin derivative, KRN5500, and its active metabolite in tumor cells.

KRN5500, (6-[4-Deoxy-4-(2E,4E)-tetradecadienoylglycyl]amino-L-glycero - beta-L-mannoheptopyranosyl]amino-9H-purine), was semi-synthesized in an attempt to increase the therapeutic effects of spicamycin analogues. The present study evaluated the antitumor activity of KRN5500 against murine tumors and human tumor xenografts. KRN5500 prolonged the survival of P388 leukemia- and B16 melanoma-bearing mice but was marginally effective on colon adenocarcinoma 26. The antitumor activity of KRN5500 (4 mg/kg/day x 5, IV) against xenografts of 10 human stomach, 14 colon and 2 esophageal cancers was evaluated with two parameters: the tumor growth inhibition rate (TGIR) and the tumor mass reduction by comparison with mitomycin C (MMC, 6.7 mg/kg/day x 1,IV). KRN5500 showed a marked efficacy in the human tumor xenograft model. The overall response rate of 26 cancers to KRN5500, evaluated by TGIR, was approximately equal to their response rate to MMC (72% vs. 73%). However, more tumors were reduced by KRN5500 than by MMC (52% vs. 39%). It is notable that the response rates of 14 colon cancers to KRN5500 were significantly higher than those to MMC, both in TGIR (69% vs. 58%) and in tumor mass reduction (46% vs. 23%). Among the tumors sensitive to KRN5500, COL-1 showed a marked response (TGIR 93%) and a significant reduction in tumor mass (0.22-fold the starting volume). In the mode of action, KRN5500 was found to show an inhibitory effect on protein synthesis in P388 cells (IC50 1.5 microM). However, KRN5500 was ineffective even at 170 microM in inhibition of protein synthesis in rabbit reticulocyte lysates.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-antitumor activity relationship of semi-synthetic spicamycin analogues.

Spicamycin, a nucleoside antibiotic containing fatty acids with a variety of chain lengths (C12-C18), showed potent antitumor activity against human gastric cancer SC-9 and human breast cancer MX-1 in a xenograft model. We have made several semi-synthetic spicamycin analogues (SPMs) which differed in the chain length of the fatty acid moiety, and examined their structure-antitumor activity relationship. The cytotoxic activities of SPMs depended on the chain length of the fatty acid moiety, with dodecanoyl, tetradecanoyl, hexadecanoyl and icosanoyl analogues (SPM VIII, SPM X, SPM XII and SPM XVI) exhibiting the most potent cytotoxic activity against P388 murine leukemia cells. SPM VIII showed the most activity against SC-9 in the human tumor xenograft model with the highest therapeutic index among SPMs. The antitumor activity of SPM VIII was superior to that of mitomycin C.