Reevaluation of immunoreactive gonadotropin-releasing hormone (GnRH) levels in general circulation in women: changes in levels and episodic patterns before, during and after gonadotropin surges

In order to reevaluate the earlier varying data regarding circulatory gonadotropin-releasing hormone (GnRH), we assayed extracted GnRH from the plasma frequently collected at mid-cycle in 11 women. For the analysis of episodic GnRH patterns and basal levels, blood samples were obtained at 6 h intervals for 72 h and at 15 min intervals for 2 h every 12 h throughout the experimental period. All blood samples were assayed for GnRH and selected samples for LH, FSH, estradiol and progesterone. For GnRH assay, 5 or 6 ml of blood was mixed with 60 mg of ethylenediaminetetraacetic acid, disodium salt, and 3 mg of phenylmethylsulfonyl floride immediately after blood collection. These enzyme inhibitors prevented the destruction of GnRH in the blood at room temperature for at least 4 h. Plasma GnRH was extracted through several steps including florisil absorption, acidic extraction and washing with organic solvent. Nonspecific immunoreactivity in the plasma was markedly decreased through this extraction process. Our assay values (approximate range, 0.1-2.0 pg/ml) of plasma GnRH in normal women corresponded to the low range of those obtained by others who used the alcohol extraction method. The basal levels of GnRH did not change significantly throughout 3 different periods, i.e., before, during and after the LH surges, and fluctuated between a small range of 0.11 and 1.44 pg/ml. Although the peak levels of GnRH observed in its episodic patterns did not change between the periods before and during the LH surges, they decreased significantly after the LH surge compared with those seen during the LH surges (0.93 +/- 0.07 vs 1.17 +/- 0.09 pg/ml, p less than 0.05). The present data demonstrate that immunoreactive GnRH in the extracted peripheral plasma does not change significantly in its mean, basal and peak levels during the periovulatory period except for a minor but significant decrease in the peak levels shortly after an LH surge.     

Evaluation of GnRH administration on the prolactin response to thyrotrophin-releasing hormone in normal women

To determine whether GnRH modifies prolactin (PRL) secretion in response to thyrotrophin-releasing hormone (TRH) in normal women, a group of eleven normal women, 23 to 40 years of age, was studied in the mid-follicular phase of the menstrual cycle. The PRL response to TRH was evaluated in serum under control conditions and after GnRH infusion. GnRH administration augmented basal PRL release and amplified TRH-induced PRL release. These results suggest that GnRH may be involved in PRL release, partly by increasing the sensitivity of the lactotrophs to TRH.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Levamisole in postoperative adjuvant immunochemotherapy for gastric cancer

Summary The usefulness of LMS in postoperative immunochemotherapy of gastric cancer was investigated. In compliance with the protocol, MMC was given at a dose of 20 mg on the day of gastrectomy, and an additional 10 mg on the next day IV. The patients receiving 600 mg Tegafur daily were then divided into two groups according to whether LMS was also given or not. LMS was administered for 3 days before the operation in a daily dose of 150 mg and for 1 year or more after operation according to a schedule of 3 days' administration followed by an 11-day interval. The 2-year follow-up demonstrated that in stage III patients, the LMS (+) regimen was superior to the LMS (–) regimen, since the former prolonged the relapse-free interval significantly. The survival rate for stage III disease was also significantly higher in the LMS (+) than in the LMS (–) group. There was no significant difference in the incidence of subjective or objective side-effects between two groups. The incidence of agranulocytosis was comparable in the two groups.Gastrointestinal Cancer Research Group, Japan Levamisole Research AssociationChairmen of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationController of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationMembers of the Data Collection and Analysis SubcommitteeThis study was carried out by the Gastrointestinal Cancer Research Group, Japan LMS Research Association (directed by Prof. Kiyoshi Inokuchi, Dept. of Surgery, Kyushu University and Prof. Eiro Tsubura, Dept. of Internal Medicine, Tokushima University). The results were presented in part at the 19th General Meeting of the Japanese Society for Gastroenterological Surgery in February, 1982     

Low serum levels of FSH, LH and prolactin in luteal phase inadequacy

In order to elucidate the relationship between prolactin (PRL) levels and corpus luteum function in humans, assessment of temporal relationship between levels of PRL, LH, FSH, estradiol and progesterone was made in eleven normal cycling women and six short luteal women. All hormones were determined by specific radioimmunoassay. The mean PRL level in the luteal phase was higher than that in the follicular phase in normal women. On the other hand, no difference mean was seen between the PRL levels of follicular and luteal phases in short luteal women. In addition, follicular and luteal phase secretion of PRL in the short luteal phase (SLP) was lower than that in the normal control. LH and FSH in the follicular and luteal phases, estradiol secretion in the late follicular and early to mid-luteal phases in SLP were also lower than those in the control. These observations were consistent with the hypothesis that SLP is a sequel to aberrant folliculogenesis. In addition, it is inferred that low PRL levels in the SLP might be due to inadequate augmentation by estrogen, rather than giving PRL any positive controlling role in the maintenance of corpus luteum function.     

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Efficacy of Dietary Lipid Control in Healing High-Fat and High-Cholesterol Diet-Induced Fibrotic Steatohepatitis in Rats

Nonalcoholic steatohepatitis is related to lifestyle, particularly to dietary habits. We developed diet-induced fibrotic steatohepatitis model stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats showing steatosis, hepatic inflammation, and severe fibrosis induced by high-fat and -cholesterol (HFC) diet feeding. We aimed to clarify the efficacy of dietary intervention on the disease before and after the appearance of fibrosis. Male SHRSP5/Dmcr rats were divided into 9 groups; of these, 6 groups were fed control or HFC diet for several weeks and the remaining 3 groups represented the dietary intervention groups, which were fed the control diet after HFC diet feeding for 2 (before the appearance of fibrosis) or 8 (after the appearance of fibrosis) weeks. Dietary intervention before the appearance of fibrosis significantly improved the steatosis and reset the increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum total cholesterol (TC) levels. However, dietary intervention after the appearance of fibrosis was unable to reset the levels of hepatic TC, serum ALT, and fibrogenesis-related markers and had only a minor influence on hepatic fibrosis, although it reset the increased expression of transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). It was noted that dietary intervention improved the increased AST levels; however, aggregated CD68-positive cells were still observed around the fibrosis area, which may be related to the findings of inflammatory cytokine mRNAs. Taken together, dietary intervention for fibrotic steatohepatitis improved steatosis, although it could not completely improve fibrosis.