Specific messenger RNA expression for signal transduction molecules by lipopolysaccharide in intestinal macrophages.

Intestinal macrophages are known to display profound inflammatory anergy in response to lipopolysacchraide (LPS). To study the mechanisms of unresponsiveness of intestinal macrophages to LPS, we compared the mRNA expression of molecules associated with signal transduction of intestinal macrophages with those of other tissue macrophages. Also cellular localization of CD14 protein was examined. Intestinal, alveolar and peritoneal macrophages were isolated from rats or mice. The expression of mRNA was assessed by real-time PCR, and cellular localization of CD14 protein was examined by flow cytometry. Cellular responses to LPS were examined by production of TNF and NO. The expression of CD14 mRNA in intestinal macrophages was lower than for peritoneal macrophages but higher than for alveolar macrophages. The mRNA expression of other molecules corresponding to intracellular signal transduction in intestinal macrophages was similar with alveolar and peritoneal macrophages. Despite the presence of CD14 mRNA, proteins of CD14 were not detected on cell surfaces of intestinal macrophages, and induction of TNF or NO responding to LPS were not detected. Flow cytometric analysis demonstrated that CD14 protein was not expressed on the cell surface but was expressed inside intestinal macrophages. The unresponsiveness of intestinal macrophages after LPS exposure is considered to be largely attributed to the lack of CD14 protein on their cell surfaces. However, CD14 protein was expressed inside of the cells, suggesting that post-transcriptional regulation rather than transcriptional suppression may play a dominant role in determining the phenotype of the intestinal macrophages.

Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss.

A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNF alpha-like than TNF beta-like. The full length cDNA is 1519bp, containing a 765bp open reading frame. The gene has four exons, of 380, 49, 60 and 1030bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer.

Intradermal administration of lipopolysaccharide in treatment of human cancer.

Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m2 dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans.

Augmentation of specific tumor killing activity by tumor-infiltrating lymphocytes (TIL) in the presence of TNF-SAM2.

The effect of TNF-SAM2 on cytotoxic activity of tumor-infiltrating lymphocytes (TIL) was investigated. TIL were prepared from 11 human cancer patients. They were propagated by double in vitro stimulation with anti-CD3 monoclonal antibody and interleukin-2, and cultured for 3 weeks. The cytotoxic activity of TIL was tested with standard 4h 51Cr-release assays in the presence or the absence of TNF-SAM2. In the presence of TNF-SAM2 (500U/ml), the mean cytotoxic activity against autologous tumor cells was significantly augmented compared to that in its absence. However, the fact that cytotoxic activity against K562 and Daudi showed no difference whether substance was present or not, indicates that LAK and NK activity were not affected by TNF-SAM2. Direct cytotoxicity by exogenously added TNF-SAM2 to tumor cells was measured in 9 out of 11 cases and this revealed that cytotoxicity solely by TNF-SAM2 was seen in 3 tumors. However, there was no correlation between the augmentation of cytotoxicity by TIL in the presence of TNF-SAM2 and the cytotoxicity shown by TNF-SAM2 alone. These results suggested that TIL therapy combined with administration of exogenous TNF may exert a synergistically stronger therapeutic effect on cancer.

Enhanced production of tumour necrosis factor alpha (TNF-alpha) by its precursor on the cell surface of primed THP-1 cells.

To clarify the biological significance of tumour necrosis factor alpha (TNF-alpha) precursor, we analysed its expression at the primed and triggered stages using human monocyte-like cell line THP-1. To prime them, THP-1 cells were treated with either recombinant human interferon gamma (rIFN-gamma) or recombinant human tumour necrosis factor alpha (rTNF-alpha). At the primed stage, transient accumulation of TNF-alpha, mRNA and a small amount of 26-KDa TNF-alpha precursor was observed, and the precursor molecule was located on the cell surface. Following treatment of the primed cells with bacterial lipopolysaccharide (LPS), augmentation of transcription of TNF-alpha mRNA and production of a larger amount of TNF-alpha precursor were observed followed by secretion of a larger amount of mature TNF-alpha (17-KDa) than secreted by the unprimed cells (triggered stage). This suggests that with priming THP-1 cells might be changed to a stage where they are ready for production of a larger amount of TNF-alpha at the triggered stage. When either primed or unprimed THP-1 cells were pretreated with anti-TNF-alpha antibody, augmentation of TNF-alpha production by primed THP-1 cells was specifically suppressed, suggesting that TNF-alpha precursor itself may play an important role in the enhancement of TNF-alpha production by the primed macrophages after treatment with LPS.

A case report of recurrent cervical cancer which responded to a combination of biological therapies.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) has been challenged to human cancer patients during these years. However, as the efficacy of the therapy alone is still limited, another approach, a combination with other therapies may be required to obtain more favorable antitumor effects. Tumor necrosis factor alpha (TNF) is worth considering for combination, because at least its various potential roles in immune response suggest that it might increase the killing activity of T cells against autologous tumor cells. We attempted to treat the cancer patients with TIL combined with TNF therapy. A patient with recurrent cervical cancer was treated with multiple biological therapies including TIL and TNF therapy, and irradiation. Biological therapies consisted of endogenous and exogenous TNF (EET) therapy, TIL, administration of rTNF-SAM2 combined with hyperthermia and several biological response modifiers such as schizophyllan and lentinan. After 50 days of therapy, the recurrent tumor had remarkably decreased in size, and cystic change of the tumor was observed by CT scanning. By further continuation of these therapies, the disease condition was much improved. As these antitumor effects cannot well be explained by irradiation therapy alone, multiple biological therapies are considered to be potentially effective against the highly malignant advanced tumors.

Promotion of metastases by tumor necrosis factor in rats bearing Dunning R3327 MAT-LyLu prostatic cancer.

We examined the effects of recombinant human tumor necrosis factor (rTNF) on highly metastatic rat prostatic cancer, Dunning R3327 MAT-LyLu, in vivo. It seemed that the inhibition of tumor growth needed more than 5 x 10(5) units of rTNF and this dose was near the lethal dose. Concerning the effect of rTNF on metastasis, we found that the metastasis to lung was promoted by 1 x 10(5) units of rTNF and the promotion of metastasis was restricted by indomethacin. Although MAT-LyLu cells were producing a significant amount of prostaglandin E2, rTNF did not enhance the production. We therefore consider that the promotion of metastasis was not mediated by prostaglandin E2 from MAT-LyLu cells but was mediated by other very complicated conditions. Further studies are necessary to resolve the mechanisms.

Homeostasis as regulated by activated macrophage. V. Suppression of diabetes mellitus in non-obese diabetic mice by LPSw (a lipopolysaccharide from wheat flour).

A lipopolysaccharide from wheat flour (LPSw) which was isolated and characterized is shown to exert definitely a suppressive effect on incidence of type 1 diabetes in mice. Therapeutic effect on type 2 diabetes in patients was also obtained, though only from preliminary results. The percutaneous route of administration is most favorable. The important role that precursor tumor necrosis factor, free or cell bound, may play in this mechanism is discussed.

Homeostasis as regulated by activated macrophage. IV. Analgesic effect of LPSw, a lipopolysaccharide of wheat flour.

The effect of LPSw, a lipopolysaccharide from a water extract of wheat flour, on pain response was investigated using an acetic acid-induced writhing test in mice. LPSw inhibited writhing dose-dependently in the range of 10 ng-10 micrograms/mouse i.v. This effect reached its maximum 1.5-3 h after the LPSw inoculation and was detectable even after 8 h. The analgesic effect of LPSw was inhibited by i.v. injection of naloxone and also beta-endorphin was detected in serum and brain tissue following injection of LPSw. Preliminary clinical trials were done in which LPSw was administered percutaneously to relieve the pain of patients with herpes. The results showed that pain was relieved by this application. LPSw may be the best analgesic drug so far known, since it induces the endogenous mediator of analgesia, beta-endorphin.

Homeostasis as regulated by activated macrophage. II. LPS of plant origin other than wheat flour and their concomitant bacteria.

In order to seek a macrophage-activating substance, lipopolysaccharide (LPS) of plant origin other than that of wheat flour was surveyed. A large amount of LPS (10-100 micrograms/g) was detected in Laminaria japonica (kelp), Curcuma longa (turmeric), Undaria pinnatifida and other substances. Since concomitant bacteria possibly existing in root of farm products can be considered to contribute to LPS of plant origin, a count was taken of bacterial cells both dead and alive. This count revealed that some LPS were derived from concomitant bacteria which had probably come from root. Few concomitant bacterial cells were found in seaweed, while stem-root contained enough bacterial cells. Three predominant bacteria have been isolated and identified; Pantoea agglomerans, Enterobacter cloacae, and Serratia ficaria. These LPSs were purified and their chemical compositions were examined. They are similar to that of Escherichia coli except that their molecular sizes are smaller. Since LPS is non-toxic when taken orally or percutaneously, these LPSs may also be advantageous in the cure of intractable diseases.

Homeostasis as regulated by activated macrophage. III. Protective effect of LPSw (lipopolysaccharide (LPS) of wheat flour) on gastric ulcer in mice as compared with those of other LPS from various sources.

Protective effect of lipopolysaccharide (LPS) from various sources on gastric ulcer has been examined in mice using parenteral as well as oral route. Ulcer is induced by indomethacin, stress or alcohol. LPS was prepared from 6 species of bacteria (Escherichia coli, Pantoea agglomerans, Serratia ficaria, Enterobacter cloacae, Bordetella pertussis, Alcaligenes faecalis) and from wheat flour. When administered intravenously, LPS of Pantoea agglomerans was the most effective among other LPS examined. Lipopolysaccharide of wheat flour (LPSw) showed a significantly protective effect by the oral route, especially when given ad libitum in drinking water to mice.

Absence of a gamma-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta.

1. Complete nucleotide sequence of one of the salmon proopiomelanocortin mRNAs (POMC mRNAs) was determined. 2. The region corresponding to gamma-melanocyte-stimulating hormone (gamma-MSH) was lacking in salmon POMC mRNA, although overall organization of the multi-hormone structure was exactly the same as that of mammalian POMC mRNAs. 3. The possible evolutional history of POMC mRNA in mammalian species may be revealed from the finding of this characteristic that salmon POMC mRNA lacks the region corresponding to gamma-MSH.

Nucleotide sequence of a cloned cDNA for proopiomelanocortin precursor of chum salmon, Onchorynchus keta.

We have isolated a cDNA clone encoding salmon proopiomelanocortin precursor. Polyadenylated RNA was isolated from pituitary neurointermediate lobes and used to construct a cDNA library. The library was screened with 17 mer of oligodeoxyribonucleotides specific for the hexapeptide sequence in salmon beta-endorphin I, Phe-Met-Lys-Pro-Tyr-Thr at positions 4-9 excluding the third nucleotide. One positive clone, pSSM17 containing an insert of 1303 base pairs (bp) was characterized. Sequence determination revealed that it possessed sequences covering the entire regions encoding ACTH and beta-lipotropin and that the mRNA had the same overall organization as those of other mammalian species, i.e., the following peptide hormones were arranged in order from 5' upstream, ACTH including alpha-melanotropin and corticotropin-like intermediate lobe peptide, beta-lipotropin including gamma-lipotropin, beta-melanotropin and beta-endorphin. Amino acid sequences for putative salmon ACTH, beta-, and gamma-lipotropin were predicted. Comparison of the salmon mRNA sequence with those of mammals showed that the regions of alpha- and beta-MSH are relatively homologous, but other regions are much less so, especially in the 3' nontranslated region where it is much longer and completely heterologous.

Sequence differences between mouse embryonic y- and adult beta-globin messenger ribonucleic acids.

Poly(A)-containing RNA was isolated from yolk sac cells of BDF1 hybrid mice on the 12th day of gestation. In vitro translation of the poly(A)-containing RNA by a messenger RNA (mRNA) dependent reticulocyte lysate system revealed that this RNA contained a high concentration of mRNAs from two embryonic globin chain subunits, y-1 and y-2. Cross hybridization of complementary DNA (cDNA) to the embryonic globin mRNAs with adult reticulocyte globin mRNAs and with cloned cDNAs for adult alpha- and beta-globin chain mRNAs showed that the sequence of embryonic globin y-chain mRNA differed from that of adult beta-chain mRNA.