Short-term depletion of plasma estrogen affects hypothalamic kisspeptin-neurokinin B-dynorphin A neurons, gonadotrophs, and pulsatile luteinizing hormone secretion in female rats.

Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) regulate pulsatile luteinizing hormone (LH) secretion. These neurons express estrogen receptors and are negatively regulated by estrogen. This study aimed to determine whether estrogen supplementation after short-term ovariectomy-induced estrogen depletion has different effects on KNDy neurons depending on the timing of the supplementation. To decrease endogenous estradiol (E2) for a short time, adult female rats received a tube filled with E2 one week after ovariectomy and utilized it one week later (O1w + E). From the results of immunohistochemistry, the response to E2 was attenuated in KNDy neurons of O1w + E rats. Enlarged LH-secreting cells in the anterior pituitary were found in O1w + E rats; however, such enlarged LH cells were not found in ones without previous short-term E2 depletion. From the analysis of LH pulses, plasma LH levels were increased in O1w + E rats relative to ones without previous short-term E2 depletion. These results suggested that once endogenous sex steroids were depleted, the response to E2 in hypothalamic KNDy neurons did not fully recover in one week. Thus, short-term sex steroid depletion due to gonadectomy could alter the response to the sex steroids in KNDy neurons even though the period without sex steroids is only one week, and the alteration is likely to affect plasma hormone levels.

Intrauterine LPS inhibited arcuate Kiss1 expression, LH pulses, and ovarian function in rats.

In brief: Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. The current findings that intrauterine lipopolysaccharide is absorbed in systemic circulation and attenuates ovarian cyclic activities could provide a basis for developing novel treatments to improve fertility. Abstract: Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. Circulating lipopolysaccharide (LPS), a bacterial endotoxin causing uterine inflammation, reportedly downregulates the hypothalamic-pituitary-gonadal axis to mediate ovarian dysfunction. In contrast, the mechanism whereby intrauterine LPS affects ovarian function has not been fully clarified. This study aimed to elucidate whether uterine exposure to LPS downregulates hypothalamic kisspeptin gene (Kiss1) expression, gonadotropin release, and ovarian function. Uterine inflammation was induced by intrauterine LPS administration to ovary-intact and ovariectomized female rats. As a result, plasma LPS concentrations were substantially higher in control rats until 48 h post injection, and the estrous cyclicity was disrupted with a prolonged diestrous phase. Three days post injection, the number of Graafian follicles and plasma estradiol concentration were reduced in LPS-treated rats, while numbers of Kiss1-expressing cells in the anteroventral periventricular nucleus and arcuate nucleus (ARC) were comparable in ovary-intact rats. Four days post injection, ovulation rate and plasma progesterone levels reduced significantly while gene expression of interleukin1ß and tumor necrosis factor α was upregulated in the ovaries of LPS-treated rats that failed to ovulate. Furthermore, the number of Kiss1-expressing cells in the ARC and pulsatile luteinizing hormone (LH) release were significantly reduced in ovariectomized rats 24 h post injection. In conclusion, these results indicate that intrauterine LPS is absorbed in systemic circulation and attenuates ovarian function. This detrimental effect might be caused, at least partly, by the inhibition of ARC Kiss1 expression and LH pulses along with an induction of ovarian inflammatory response.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Morphological Analysis of the Hindbrain Glucose Sensor-Hypothalamic Neural Pathway Activated by Hindbrain Glucoprivation.

Lowered glucose availability, sensed by the hindbrain, has been suggested to enhance gluconeogenesis and food intake as well as suppress reproductive function. In fact, our previous histological and in vitro studies suggest that hindbrain ependymal cells function as a glucose sensor. The present study aimed to clarify the hindbrain glucose sensor-hypothalamic neural pathway activated in response to hindbrain glucoprivation to mediate counterregulatory physiological responses. Administration of 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, into the fourth ventricle (4V) of male rats for 0.5 hour induced messenger RNA (mRNA) expression of c-fos, a marker for cellular activation, in ependymal cells in the 4V, but not in the lateral ventricle, the third ventricle or the central canal without a significant change in blood glucose and testosterone levels. Administration of 2DG into the 4V for 1 hour significantly increased blood glucose levels, food intake, and decreased blood testosterone levels. Simultaneously, the expression of c-Fos protein was detected in the 4V ependymal cells; dopamine ß-hydroxylase-immunoreactive cells in the C1, C2, and A6 regions; neuropeptide Y (NPY) mRNA-positive cells in the C2; corticotropin-releasing hormone (CRH) mRNA-positive cells in the hypothalamic paraventricular nucleus (PVN); and NPY mRNA-positive cells in the arcuate nucleus (ARC). Taken together, these results suggest that lowered glucose availability, sensed by 4V ependymal cells, activates hindbrain catecholaminergic and/or NPY neurons followed by CRH neurons in the PVN and NPY neurons in the ARC, thereby leading to counterregulatory responses, such as an enhancement of gluconeogenesis, increased food intake, and suppression of sex steroid secretion.

Reduction of arcuate kappa-opioid receptor-expressing cells increased luteinizing hormone pulse frequency in female rats.

The brain mechanism responsible for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) is important for maintaining reproductive function in mammals. Accumulating evidence suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH and subsequent gonadotropin secretion. Dynorphin A (Dyn) and its receptor, kappa-opioid receptor (KOR, encoded by Oprk1), have been shown to be involved in the suppression of pulsatile GnRH/luteinizing hormone (LH) release. On the other hand, it is still unclear whether the inhibitory Dyn signaling affects KNDy neurons or KOR-expressing non-KNDy cells in the ARC or other brain regions. We therefore aimed to clarify the role of ARC-specific Dyn-KOR signaling in the regulation of pulsatile GnRH/LH release by the ARC specific cell deletion of KOR-expressing cells using Dyn-conjugated-saporin (Dyn-SAP). Estrogen-primed ovariectomized female rats were administered Dyn-SAP to the ARC. In situ hybridization of Oprk1 showed that ARC Dyn-SAP administration significantly decreased the number of Oprk1-expressing cells in the ARC, but not in the ventromedial hypothalamic nucleus and paraventricular nucleus. The frequency of LH pulses significantly increased in animals bearing the ARC Dyn-SAP administration. The number of Kiss1-expressing cells in the ARC was not affected by ARC Dyn-SAP treatment. Dyn-KOR signaling within the ARC seems to mediate the suppression of the frequency of pulsatile GnRH/LH release, and ARC non-KNDy KOR neurons may be involved in the mechanism modulating GnRH/LH pulse generation.

Inducible Kiss1 knockdown in the hypothalamic arcuate nucleus suppressed pulsatile secretion of luteinizing hormone in male mice.

Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

Role of kisspeptin neurons as a GnRH surge generator: Comparative aspects in rodents and non-rodent mammals.

Ovulation is an essential phenomenon for reproduction in mammalian females along with follicular growth. It is well established that gonadal function is controlled by the neuroendocrine system called the hypothalamus-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons, localized in the hypothalamus, had been considered to be the head in governing the HPG axis for a long time until the discovery of kisspeptin. In females, induction of ovulation and folliculogenesis has been linked to a surge mode and pulse mode of GnRH releases, respectively. The mechanisms of how the two modes of GnRH are differently regulated had long remained elusive. The discovery of kisspeptin neurons, distributed in two hypothalamic nuclei, such as the arcuate nucleus in the caudal hypothalamus and preoptic area or the anteroventral periventricular nucleus in the rostral hypothalamic regions, and analyses of the detailed functions of kisspeptin neurons have led marked progress on the understanding of different mechanisms regulating GnRH surges (ovulation) and GnRH pulses (folliculogenesis). The present review will focus on the role of kisspeptin neurons as the GnRH surge generator, including the sexual differentiation of the surge generation system and factors that regulate the surge generator. Comparative aspects between mammalian species are especially focused on.

Neonatal Estrogen Causes Irreversible Male Infertility via Specific Suppressive Action on Hypothalamic Kiss1 Neurons.

Aberrant exposure to estrogen-like compounds during the critical developmental period may cause improper hypothalamic programming, thus resulting in reproductive dysfunction in adulthood in male mammals. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) have been suggested to govern tonic GnRH/gonadotropin release to control reproduction in male mammals. In this study, we report that chronic exposure to supraphysiological levels of estrogen during the neonatal period caused an irreversible suppression of KNDy genes in the ARC, resulting in reproductive dysfunction in male rats. Daily estradiol benzoate (EB) administration from days 0 to 10 postpartum caused smaller seminiferous tubules, abnormal spermatogenesis, and a decrease in plasma testosterone in adult male rats. The neonatal EB treatment profoundly suppressed LH pulse and ARC KNDy gene expression at adulthood, but it failed to affect the number of GnRH gene-expressing cells in male rats. The EB treatment failed to affect gene expression of other neuropeptides, such as GHRH, proopiomelanocortin, and agouti-related protein in the ARC, suggesting that ARC KNDy neurons would be a specific target of neonatal estrogen to cause male reproductive dysfunction. Because LH secretory responses to kisspeptin challenge and GnRH expression were spared in male rats with the EB treatment, LH pulse suppression is most probably due to ARC KNDy deficiency. Taken together, the current study indicates that chronic exposure to estrogenic chemicals in the developing brain causes a defect of ARC KNDy neurons, resulting in an inhibition of pulsatile GnRH/LH release and the failure of spermatogenesis and steroidogenesis.

Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

Milk fat globule epidermal growth factor-8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms.

Carcinogenesis reflects the dynamic interplay of transformed cells and normal host elements, but cancer treatments typically target each compartment separately. Within the tumor microenvironment, the secreted protein milk fat globule epidermal growth factor-8 (MFG-E8) stimulates disease progression through coordinated alpha(v)beta(3) integrin signaling in tumor and host cells. MFG-E8 enhances tumor cell survival, invasion, and angiogenesis, and contributes to local immune suppression. We show that systemic MFG-E8 blockade cooperates with cytotoxic chemotherapy, molecularly targeted therapy, and radiation therapy to induce destruction of various types of established mouse tumors. The combination treatments evoke extensive tumor cell apoptosis that is coupled to efficient dendritic cell cross-presentation of dying tumor cells. This linkage engenders potent antitumor effector T cells but inhibits FoxP3(+) T reg cells, thereby achieving long-term protective immunity. Collectively, these findings suggest that systemic MFG-E8 blockade might intensify the antitumor activities of existing therapeutic regimens through coordinated cell-autonomous and immune-mediated mechanisms.

Combined mobilization and stimulation of tumor-infiltrating dendritic cells and natural killer cells with Flt3 ligand and IL-18 in vivo induces systemic antitumor immunity.

It was hypothesized that if dendritic cells (DC) could be efficiently manipulated in vivo, this might enable functional maturation and retention of their potent functions and might represent a more promising approach in DC immunotherapy. The present study focused on the modulation of DC in tumor microenvironment using Fms-like thyrosine kinase 3 ligand (Flt3L) combined with interferon-gamma-inducing factor (IL-18). Tumor-inoculated mice were treated with in vivo electroporation (IVE) of expression plasmids carrying complementary DNA of Flt3L. As a combination therapy, mice in the other group were treated with intra-tumoral injection of adenoviral vector carrying IL-18 gene (Ad.IL-18). Significant antitumor effect was observed in mice treated with Ad.IL-18 alone when compared with that of control (P < 0.01). Complete eradication was observed more frequently (100%versus 33%: P < 0.05) in the mice treated with Flt3L and Ad.IL-18 when compared with the mice treated with Ad.IL-18 alone. In un-injected distant tumor, significant antitumor responses were observed only in the mice treated with combination therapy. Lymphoid cells in lymph nodes of mice treated with combination therapy showed significant cytolytic activity against inoculated tumor cells and YAC-1 cells when compared with the lymphoid cells in other groups. In the tumor microenvironment, combination therapy resulted in the recruitment of mobilized DC into the tumor bed, although Flt3L-IVE alone had an effect in the peri-tumoral area. Tumor-infiltrating DC in mice treated with combination therapy showed higher CD86 expression and more potent allogeneic T-cell stimulatory capacity. These results may suggest that local expression of IL-18 combined with in vivo DC mobilization with Flt3L is clinically applicable as a new strategy of DC immunotherapy.

Systemic administration of IL-23 induces potent antitumor immunity primarily mediated through Th1-type response in association with the endogenously expressed IL-12.

IL-23, a cytokine, which is composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit, has been shown to preferentially act on Th1 effector/memory CD4+ T cells and to induce their proliferation and IFN-gamma production. The IL-23 is also reported to act on Th17-CD4+ T cells, which are involved in inducing tissue injury. In this study, we examined the antitumor effects associated with systemic administration of IL-23 and their mechanisms in mouse tumor system. Systemic administration of high-dose IL-23 was achieved using in vivo electroporation of IL-23 plasmid DNA into the pretibial muscles of C57BL/6 mice. The IL-23 treatment was associated with significant suppression of the growth of pre-existing MCA205 fibrosarcoma and prolongation of the survival of treated mice without significant toxicity when compared with those of the mice treated with EGFP. Although the therapeutic outcomes were similar to those with the IL-12 treatment, the IL-23 treatment induced characteristic immune responses distinctive to those of IL-12 treatment. The IL-23 administration even at the therapeutic levels did not induce detectable IFN-gamma concentration in the serum. In vivo depletion of CD4+ T cells, CD8+ T cells, or NK cells significantly inhibited the antitumor effects of IL-23. Furthermore, the CD4+ T cells in the lymph nodes in the IL-23-treated mice showed significant IFN-gamma and IL-17 response upon anti-CD3 mAb stimulation in vitro. These results and the ones in the IFN-gamma or IL-12 gene knockout mice suggest that potent antitumor effects of IL-23 treatment could be achieved when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12.

Generation of mature dendritic cells fully capable of T helper type 1 polarization using OK-432 combined with prostaglandin E(2).

Dendritic cell (DC) administration appears to be a very promising approach for the immunotherapy of cancer. The results of clinical studies have suggested that the nature and the magnitude of antitumor immune responses are critically affected by DC functions, including production of T helper type 1 (Th1)-inducing cytokines, activation of T cell subsets and natural killer (NK) cells, and migration from peripheral tissues to the T cell area of the draining lymph nodes. Administration of immature DCs could fail to fully stimulate antigen-specific immune responses and might induce tolerance under some conditions. In this study, we developed a method to obtain fully mature DCs, and we compared in detail the DCs thus obtained with those obtained using a maturation stimulus termed monocyte-derived medium (MCM)-mimic, which is a mixture of recombinant cytokines and prostaglandin E(2) (PGE(2)) mimicking the components of monocyte-conditioned medium. Using DCs derived from monocytes of advanced cancer patients in this study, we found that DCs stimulated with OK-432 alone showed phenotypes similar to those of mature DCs induced using MCM-mimic, though with better secretion of IL-6 and IL-12. However, these DCs were found to have poor migratory capacity associated with the marginal expression of CCR7. When OK-432 was combined with PGE(2), the CCR7 expression and migratory capacity of DCs were significantly improved without impairing other immuno-stimulatory functions. These results suggest that stimulation with the combination of OK-432 and PGE(2) could be applicable as an alternative to MCM-mimic in clinical trials which require fully matured DCs to induce Th1-type immune responses against tumor cells even in patients with advanced cancer.

A novel tumor-vaccine cell therapy using bone marrow-derived dendritic cell type 1 and antigen-specific Th1 cells.

Dendritic cell (DC)-based tumor-vaccine therapy is a rational strategy for tumor immunotherapy. However, using this protocol, it is still difficult to induce long-term regression in established tumor-bearing mice. To overcome this problem we developed a novel tumor-vaccine therapy, combining inactivated tumor cells with bone marrow-derived DC type 1 (BMDC1) and antigen-specific T(h)1 cells. BALB/c mice were intradermally inoculated with A20-OVA tumor cells expressing ovalbumin (OVA) as a model tumor antigen. After A20-OVA tumor mass became palpable (6-8 mm), mice were treated with DC-based vaccine therapy in various protocols. A complete cure of tumor-bearing mice was induced only when mice were repeatedly vaccinated with inactivated A20-OVA cells, OVA-pulsed BMDC1 and OVA-specific T(h)1 cells. Regression of tumor cells was associated with induction of T(h)1/T(c)1-dominant antitumor immunity. Removal of one of these cellular components during vaccination resulted in failure to completely cure tumor-bearing mice. Moreover, BMDC2 cells could not replace the therapeutic effect of BMDC1 cells combined with T(h)1 cells. Thus, we propose a novel tumor-vaccine cell therapy using DC1 and T(h)1 cells.