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Human protection policies require favorable risk–benefit judgments prior to launch of clinical trials. For phase I and II trials, evidence for such judgment often stems from preclinical efficacy studies (PCESs). We undertook a systematic investigation of application materials (investigator brochures [IBs]) presented for ethics review for phase I and II trials to assess the content and properties of PCESs contained in them. Using a sample of 109 IBs most recently approved at 3 institutional review boards based at German Medical Faculties between the years 2010–2016, we identified 708 unique PCESs. We then rated all identified PCESs for their reporting on study elements that help to address validity threats, whether they referenced published reports, and the direction of their results. Altogether, the 109 IBs reported on 708 PCESs. Less than 5% of all PCESs described elements essential for reducing validity threats such as randomization, sample size calculation, and blinded outcome assessment. For most PCESs (89%), no reference to a published report was provided. Only 6% of all PCESs reported an outcome demonstrating no effect. For the majority of IBs (82%), all PCESs were described as reporting positive findings. Our results show that most IBs for phase I/II studies did not allow evaluators to systematically appraise the strength of the supporting preclinical findings. The very rare reporting of PCESs that demonstrated no effect raises concerns about potential design or reporting biases. Poor PCES design and reporting thwart risk–benefit evaluation during ethical review of phase I/II studies., Author summary To make a clinical trial ethical, regulatory agencies and institutional review boards have to judge whether the trial-related benefits (the knowledge gain) outweigh the trial-inherent risks. For early-phase human research, these risk–benefit assessments are often based on evidence from preclinical animal studies reported in so-called “investigator brochures.” However, our analysis shows that the vast majority of such investigator brochures lack sufficient information to systematically appraise the strength of the supporting preclinical findings. Furthermore, the very rare reporting of preclinical efficacy studies that demonstrated no effect raises concerns about potential design and/or reporting biases. The poor preclinical study design and reporting thwarts risk–benefit evaluation during ethical review of early human research. Regulators should develop standards for the design and reporting of preclinical efficacy studies in order to support the conduct of ethical clinical trials.

Modern approaches for research with human biospecimens employ a variety of substantially different types of ethics approval and informed consent. In most cases, standard ethics reporting such as “consent and approval was obtained” is no longer meaningful. A structured analysis of 120 biospecimen studies recently published in top journals revealed that more than 85% reported on consent and approval, but in more than 90% of cases, this reporting was insufficient and thus potentially misleading. Editorial policies, reporting guidelines, and material transfer agreements should include recommendations for meaningful ethics reporting in biospecimen research. Meaningful ethics reporting is possible without higher word counts and could support public trust as well as networked research., This Perspective shows that while ethics reporting on consent and approval for biospecimen research is widespread, for the majority of cases it is not meaningful; the authors make pragmatic suggestions of substantial changes needed to remedy this.

Vitamin D receptor (VDR) agonists are currently the agents of choice for the treatment of psoriasis, a skin inflammatory indication that is believed to involve an autoimmune component. 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D, has shown efficacy in animal autoimmune disease models of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and type I diabetes. However, the side effect of 1,25-(OH)2D3and its synthetic secosteroidal analogs is hypercalcemia, which is a major impediment in their clinical development for autoimmune diseases. Hypercalcemia develops as a result of the action of VDR agonists on the intestine. Here, we describe the identification of a VDR modulator (VDRM) compound A that was transcriptionally less active in intestinal cells and as a result exhibited less calcemic activityin vivothan 1,25-(OH)2D3. Cytokine analysis indicated that the VDRM not only modulated the T-helper cell balance from Th1 to Th2 effector function but also inhibited Th17 differentiation. Finally, we demonstrate that the oral administration of compound A inhibited the induction and progress of experimental autoimmune encephalomyelitis in mice without causing hypercalcemia.

Nuclear receptors are members of a superfamily of DNA-binding transcriptional factors. Hormone-induced gene activation by nuclear receptors involves a variety of biological phenomena such as cell proliferation, differentiation, and development (31, 33). A central question in the field is how a single nuclear receptor molecule elicits complex responses of gene activation or repression in response to a hormonal stimulus in a cell-specific manner. There is evidence that tissue selectivity of gene expression induced by liganded receptors involves the coordination and assembly of an array of coregulatory proteins (9, 15, 26, 33, 53). Together with nuclear receptors, these cofactors are regulated at multiple levels, including tissue-specific distribution (22, 41, 54), variation of the assembly on each subset of hormone response elements (57), and cell-specific interactions between the nuclear receptors and their cofactors (33, 36, 54). Members of the nuclear receptor family share several structurally related domains (31). The N-terminal transactivation regions of the receptors have variable primary sequences and are largely responsible for the functional differences among the receptor isoforms. The DNA-binding domain (DBD), which targets the receptors to their cognate hormone response elements, is composed of two conserved zinc fingers (31). The C-terminal ligand-binding domain (LBD) is responsible for binding to ligand and enables ligand-dependent disassociation of corepressor and association with coactivators (9). A common LXXLL motif present in most coactivators provides the interface for interaction between conserved LBD and coactivator molecules, including CBP/p300 (24), the SRC-1 family (26, 37), PGC-1 (22, 35, 41), CIA (48), and TRBP/ASC-2/AIB3/RAP250/PRIP/NRC (11, 23, 25, 30, 58). However, whether selective interaction between various nuclear receptors and coactivators occurs to mediate cell-specific action remains to be elucidated (54). Protein cofactors specialized for interaction with the DBD of nuclear receptors are less characterized or defined. The DBD is the most highly conserved domain among members of the nuclear receptor superfamily (31). It plays an important role in determining the selectivity of DNA binding as well as directing the dimerization of nuclear receptors (6). Extensive studies of the structure and function of the DBD have revealed that receptor dimerization and binding of the DBD to each hormone response element rely on the variation of primary sequences around its zinc fingers and, in turn, contribute to its selectivity (6, 57). However, much less is known about regulation by nuclear cofactors that bind to the DBD. Several cofactors that interact with the DBDs of nuclear receptors have been reported, including SNURF. SNURF is characterized as a small nuclear RING finger protein that activates androgen receptor (AR)-mediated transcription by interaction with the DBD of AR (34, 39). Interestingly, SNURF facilitates nuclear trafficking of AR in a ligand-dependent manner (40). Several other protein factors, including ANPK, ARIP3, and ARIP4, have also been suggested to bind to the AR DBD and to modulate AR-mediated transactivation (21). In addition, DNA-dependent protein kinase was shown to interact with the DBD of progesterone receptor (PR) (45). The DBD of thyroid hormone receptor (TR) was suggested to interact with cofactors with repressive function (28). A recent report also indicates that the DBD of TR and retinoid X receptor (RXR) associates with PSF, a corepressor that recruits Sin3A to the receptor DBD (32). Another group of proteins that influence DNA binding of nuclear receptors is the high-mobility-group (HMG) proteins (7, 8, 38, 43). In addition to facilitating the binding of nuclear receptors to DNA, HMG proteins have diverse regulatory functions in DNA binding and chromatin architecture (16). The DBDs of nuclear receptors have also been shown to interact with other transcriptional factors such as NF-κB and AP-1, with resultant regulation of transcriptional repression (51). Since the DBDs of nuclear receptors are evolutionarily conserved, DBD-binding coactivators might also retain other conserved functions in the action of various DBDs of nuclear receptors. Nuclear receptor-mediated target gene expression is largely tissue and cell specific, and it is subject to coordinate regulation by cytoplasmic signaling (31). Compared to the extensive studies of the nuclear function of nuclear receptors and cofactors, the regulation of these cofactors by signaling pathways, including phosphorylation, is less clear. However, the cytoplasmic hormonal effects of nuclear receptor ligands, now also referred to as rapid or nongenomic effects, have long been observed in cells. For example, thyroid hormone stimulates the epidermal growth factor pathway (27), estrogen induces mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase signaling (29), and tamoxifen affects insulin-like growth factor I receptor action in breast cancer cells (17). In addition to binding of the receptor LBD in the nucleus, ligands may thus stimulate various signaling molecules and ultimately modify protein factors, including the coactivators that are important for nuclear receptor action. In this regard, the regulation of cofactors by hormones via kinase signaling might also contribute to a large extent of the cell-specific effects. Several lines of evidence have supported the notion that nuclear cofactors are kinase regulated. These include phosphorylation of p300 by protein kinase C (PKC) at Ser89 with inhibition of its transactivation (56), phosphorylation and regulation of SRC-1 by MAPK via protein kinase A (PKA) signaling (10), and phosphorylation of TRBP by DNA-dependent protein kinase (23). Thus, posttranslational modification of cofactors may be a common mechanism to mediate, at least in part, the specificity and selectivity of nuclear receptors in transcriptional regulation. We report here the isolation and characterization of a tissue-specific nuclear receptor coactivator, GT198. GT198 contains a leucine dimerization domain and interacts with nuclear receptors via the DBD. GT198 enhances nuclear receptor-mediated transcription, and coactivation by GT198 may be subject to kinase regulation. The mechanism of its activation is distinguishable from LBD-interacting coactivators such as SRC-1, TRBP, and CBP. GT198 represents a new class of DBD-interacting nuclear receptor coactivators.

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes. Controversy exists, however, concerning whether ER has a role outside the nucleus, particularly in mediating the cardiovascular protective effects of oestrogen. Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ER alpha are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ER alpha with PI(3)K.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate synthesis and release of luteinizing hormone (LH). GnRH is secreted intermittently by the hypothalamus, producing pulsatile LH release, and a pulsatile GnRH stimulus is required to maintain LH secretion. We report the regulatory effects of GnRH pulse injections on pituitary concentrations of LH alpha and beta subunit mRNAs in a castrated/testosterone-replaced male rat model. Replacement with physiologic amounts of testosterone decreased concentrations of both LH subunit mRNAs. GnRH pulse injections (10-250 ng per pulse given every 30 min for 48 hr) increased both mRNA concentrations, but the dose response patterns were markedly different. alpha subunit mRNA was increased by all GnRH doses but not the levels seen after castration alone. In contrast, LH beta subunit mRNA concentrations showed a marked dependence on GnRH dose. Maximal responses, to values similar to those in castrates, occurred after 25-ng GnRH pulses, and larger doses produced a smaller increase in LH beta subunit mRNA. Both the acute LH secretory response to GnRH and the number of GnRH receptors followed a pattern similar to the LH beta subunit mRNA concentration and were maximal after the 25-ng GnRH dose. These results show that GnRH can differentially regulate LH subunit mRNAs and suggest that concentrations of LH beta subunit mRNA may be a limiting factor in GnRH-stimulated LH release.

In our studies on the molecular biology of human gastrin-releasing peptide (GRP), we have discovered an example of a change in translational reading frame apparently produced through alternative RNA splicing. Complementary DNAs prepared from a pulmonary carcinoid tumor rich in GRP immunoreactivity had one of two different-sized internal DNA fragments after digestion with the restriction enzyme Pvu II. Nucleotide sequences of the two DNA fragments were identical except for 19 additional nucleotides present in the larger fragment. The region of the mRNA containing the 19 nucleotides corresponded to the carboxyl-terminal region of the human GRP precursor. The resulting shift in reading frame causes a difference of 10 amino acids in size and an overall sequence difference of 27 amino acids between the two GRP prohormones so formed. The change in reading frame described here is unusual in eukaryotes and is yet another mechanism to produce diversity in the generation of biological peptides.

In the course of studies of cell-mediated immunity in Graves' disease, we noted that normal peripheral blood monocytes, when stimulated by bacterial lipopolysaccharide, conditioned their media with a factor that had the physicochemical properties of the lymphokine interleukin-1 (IL-1) and that enhanced DNA synthesis and replication in quiescent FRTL5 cells, a line of nontransformed rat thyroid follicular cells. This finding led to the present studies, in which the effect of IL-1 (recombinant IL-1-p) on DNA synthesis in FRTL5 was explored. In the absence of serum, IL-1 induced a small, but significant, increase in [3H]thymidine incorporation into DNA. Calf serum (0.5\%) alone also stimulated DNA synthesis slightly, but it greatly enhanced, in a synergistic manner, the stimulatory response to IL-1, decreasing the minimally effective concentration of IL-1 and amplifying the response to higher concentrations. A similar synergism was noted when quiescent FRTL5 were cultured with a combination of IL-1 and a low concentration of insulin-like growth factor-I (IGF-I), which itself stimulated DNA synthesis modestly. IL-1 also increased levels of the mRNA of the proto-oncogene c-myc in quiescent FRTL5, as TSH does, an effect thought to reflect commitment of the cell to increased growth. The findings indicate that IL-1 is an independent stimulator of thyroid cell growth, and that its effects are greatly enhanced by serum, probably in large measure by the IGF-I contained therein. They raise the possibility that IL-1 generated locally by intrathyroid macrophages may act directly by a short-loop mechanism to increase goiter formation in autoimmune thyroid disease.

Galanin is a peptide widely distributed throughout vertebrate central and peripheral nervous systems. Although its precise physiologic role is unknown, it can stimulate the pituitary secretion of prolactin and growth hormone. We examined the control of rat galanin (rGal) gene expression in the anterior pituitary using RNA blot and in situ hybridization analyses and using specific RIA. Pituitaries of normal male and ovariectomized female rats contained little detectable rGal mRNA. Treatment of these animals with 17 beta-estradiol increased pituitary rGal mRNA up to 4000-fold. These increases depended on time and dose of estrogen administration and correlated with up to 50-fold increases in pituitary galanin-like immunoreactivity. Galanin-like immunoreactivity was detectable in the plasma of estrogen-treated animals. Pituitary levels of rGal mRNA in female rats varied greater than 30-fold during the estrous cycle, with a peak on estrus and a nadir on diestrus. Estrogen-induced rGal gene expression was also observed in transplantable MtTW15 prolactin- and growth hormone-containing tumors but not in neuronal tissues expressing this gene. These data demonstrate that rGal is a secreted product of rat anterior pituitary cells, where its gene expression is strongly affected by physiologic levels of circulating estrogen.