Review of Relative effectiveness assessments (REAs) of pharmaceuticals at the European network for health technology assessment (EUnetHTA): A first step towards a consolidated European perspective on comparative effectiveness & safety?

OBJECTIVES: REAs from Joint Action (JA1-3) were reviewed and compared versus Health Technology Assessments (HTA) in France, Germany, UK, Italy. METHODS: EUnetHTA REAs published until end of 2019 were identified. Leveraging information derived from the HTA bodies' website key process (population; timing; national HTA bodies involved) and content characteristics (evidence base; comparative therapy, endpoints, subgroups) were determined and compared against national appraisals. RESULTS: All twelve pharmaceutical EUnetHTA assessment finalized until end of 2019 were included with Ustekinumab being the most recent (October 2019) and Pazopanib the first assessment (September 2012). In all but three assessments EUnetHTA's assessment did not cover the full EMA indication. Since JA3 time intervals between EMA approval and EUnetHTA assessment were < 80 days. Number of (co-)authoring HTA bodies ranged between 2 (in 6 REAs) and > 10 (Pazopanib). EUnetHTA did consider non - RCT evidence in 7 procedures; take a rather inclusive approach regarding appropriate comparative treatments; approach endpoints less restrictively than e.g. the German IQWiG/GBA; not apply a predetermined set of subgroups analyses. In seven REAs, national appraisal showed inhomogeneities across the 4 countries. National appraisals for Sotagliflozin and Ustekinumab were not yet available. CONCLUSIONS: A joint European HTA assessment has the potential to address the challenge of heterogeneity across the various national European HTA bodies and to determine joint European clinical development data standards that are aligned with regulatory requirements.

Control of androgen cytosol receptor concentrations in Sertoli cells: effect of androgens.

Androgen receptor concentrations were measured in Sertoli cells under a variety of conditions. After brief (30- to 60-min) incubation of cultured cells with [3H]R1881 (methyltrienolone), cytosol receptor concentrations were greatly diminished, and nuclear bound steroid was elevated. Removal of the exogenous steroid was accompanied by a return of cytosol receptor to preincubation concentrations by 1 h and a slower decline in nuclear bound steroid. In the continued presence of R1881 or testosterone, cytoplasmic receptor concentrations declined and then returned to or above preincubation concentrations by 6-17 h. Actinomycin-D and cycloheximide did not alter this pattern. Over this same interval, nuclear bound steroid concentrations remained elevated. Exposure of the cells to R1881 or testosterone for the entire 72-h culture period did not alter cytoplasmic receptor concentrations. Cytoplasmic androgen receptor concentrations were decreased in Sertoli cells from hypophysectomized rats 15-22 days after surgery compared to those in cells from intact controls. Treatment with testosterone propionate (0.5 mg/day) or FSH (75 micrograms/day) prevented the decline in receptor concentrations. Cryptorchidy (33 days) also decreased cytosol receptor concentrations. These data indicate that exposure to androgens can influence Sertoli cell cytosol androgen receptor concentrations in vitro and in vivo. The Sertoli cell adapts to the continual presence of androgens with a return of cytosol receptor while maintaining elevated concentrations of nuclear bound steroid. In vivo, androgen treatment can maintain cytoplasmic receptor concentrations in the absence of pituitary hormones in the immature rat.

Maturational and hormonal influences on Sertoli cell function.

The maturation of Sertoli cell function was studied by observing properties of cells obtained from 15-, 25-, and 35-day-old rats after 3 days in culture gamma-Glutamyl transpeptidase activity, expressed per mg DNA or per mg protein, and total and soluble protein to DNA ratios increased with age. In contrast, lactate dehydrogenase activity was unchanged between 15 and 35 days of age. Secreted protein to DNA ratios and the secretion of lactate, androgen-binding protein (ABP), and transferrin per mg DNA also increased with age. Two-dimensional electrophoresis maps of [35S]methionine-labeled secretory proteins indicated the appearance of two specific bands [mol wt, 66,000; pI 6.0-6.8 (band 1); mol wt, 56,000; pI 5.3-6.0 (band 2)] between 15 and 35 days of age. The hormone dependence of these parameters was studied in Sertoli cells isolated from rats hypophysectomized at 20 days of age and subsequently treated with oil, testosterone propionate, or testosterone propionate plus FSH for 15-21 days. Hypophysectomy decreased total, soluble, and secreted protein to DNA ratios; hormone treatment in vivo increased these ratios compared to oil treatment. gamma-Glutamyl transpeptidase activity was significantly decreased by hypophysectomy and increased, compared to oil treatment, by hormone treatment. In contrast, lactate dehydrogenase activity per mg DNA was also decreased by hypophysectomy, but was unaffected by hormone treatment. [35S]Methionine incorporation into secreted protein and secretion of ABP and lactate per mg DNA were all decreased by hypophysectomy, whereas transferrin secretion per mg DNA was unaffected. While the hormone treatments increased ABP secretion, they had no effect on lactate or transferrin secretion, expressed per mg DNA. The [35S]methionine-labeled secretory proteins (bands 1 and 2) were not visible in two-dimensional electrophoresis maps after hypophysectomy of the donor animals. Treatment with testosterone propionate or testosterone propionate plus FSH in vivo resulted in the appearance of the acidic components of band 2. These data demonstrate that changes reflecting in vivo maturational patterns and hormonal influences on Sertoli cell function persist, at least in a relative sense, after 3 days in culture. Although the majority of [35S]methionine-labeled Sertoli cell cellular and secretory proteins were present under all conditions, maturation- and hormone-dependent changes in a number of specific functions were demonstrated.

The role of the chief marketing executive in hospitals. A survey.

The role and support for the Chief Marketing Executive (CME) in hospitals is studies through a 97 hospital survey. The authors found that the majority of CMEs report at a high level in the organization, but their staffs are still small and their duties diverse. However, the budgets for advertising and market research are increasing. The authors anticipate the CME's role to continue evolving into a strategist as hospitals define more clearly the CME's duties and recognize marketing's contribution to the organization.

The chiropractic market segment: a viable market opportunity for M.D.'s?

Physicians have traditionally paid closer attention to the competition from other medical practitioners, but have ignored competition arising from "alternative therapies" such as hypnosis, acupuncture, or chiropractic. The purpose of this article is to survey the market segment served by one such unconventional practitioner, i.e., chiropractors, with the intention of determining its likelihood as a viable market to be pursued by M.D.'s.

Interaction of progestins with Sertoli cell androgen receptors.

In an attempt to distinguish between possible androgen- and progestin-mediated mechanisms in the Sertoli cell, two steroids previously shown to have high affinity for both progesterone and androgen receptors (U13851, 17 alpha-ethynyl-17-hydroxy-7 alpha-methylestr-4-en-3-one and U6817, 17-hydroxy-19-nor-17 alpha-pregn-4-en-3-one) have been compared with respect to a number of parameters with two steroids having high affinity only for progesterone receptors (U49836, 17 beta-methoxyestr-4-en-3-one and U56902, 17-methoxy-17 alpha-pregn-4-3n-20-yn-3-one). U13851 and U6817 were the most potent competitors for cytosol [3H]-R1881 (17-beta-hydroxy-17-methylestra-4,9,11-triene-3-one) binding sites and nuclear accumulation of label. While U49836 and U56902 were considerably less effective as competitors against [3H]-R1881, they were more effective than U13851 and U6817 as competitors against [3H]-progesterone. Only U13851 and R1881 increased Sertoli cell nuclear RNA polymerase II activity. These data substantiate previous suggestions that progestin binding proteins distinct from androgen receptors may exist in Sertoli cells. Moreover, the data also suggest that progestins which stimulate RNA polymerase II activity do so via androgenic mechanisms.

Nuclear deprimerones (low molecular weight peptides controlling gene expression) are associated with DNA and nuclear RNA.

Isolated, deproteinized nucleic acids from calf thymus and rat liver nuclei were fractionated on Bio-Gel A-5m into DNA and RNA fractions. Extraction of deproteinized DNA and nuclear RNA with 80% ethanol at pH 9.5 yielded low molecular weight peptides (deprimerones) capable of inhibiting DNA transcription in a cell-free system. The extracted peptidic fractions were further fractionated on a Sephadex G-25 column and collected as a fraction of mol. wt. between 1 500 and 600. The yield of DNA deprimerones was about 30 microgram/mg DNA and of nuclear RNA deprimerones 150-200 microgram/mg RNA (measured by amino acid composition). The nucleoplasm contained a negligible amount of free, soluble peptides (about 18 microgram/g tissue). The DNA and RNA deprimerones were characterized by their amino acid composition and by two-dimensional thin layer chromatography on cellulose gel plates which yielded two major and two minor fractions. DNA deprimerones isolated in this way are very similar, if not identical, to the affinity DNA deprimerones isolated from a nuclear extract on DNA-cellulose but differ from RNA deprimerones. DNA and RNA deprimerones were also compared with the nuclear deprimerones extracted directly from the lysed nuclei. Their amino acid composition is different from both DNA and nuclear RNA deprimerones. Two-dimensional thin layer chromatography on cellulose plates yielded about ten fractions (spots) with various amino acid composition and inhibitory activity for cell-free transcription.

Effect of hypotonic treatment on Sertoli cell purity and function in culture.

Commonly used enzymic methods for the isolation of rat Sertoli cells yield populations containing approximately 15% germ cells. Although the germ cells become eliminated after several media changes, they could interfere with the use of Sertoli cells for critical studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4) was found to eliminate most of the residual contaminating germ cells. The duration of this treatment varied from 1.0 to 10 min, depending on cell density in the culture, the degree of germ cell contamination, and the age of animals used for Sertoli cell isolation. In a study of 95% pure, 7-d Sertoli cell cultures, the hypotonic treatment did not alter the DNA or RNA content per dish or the incorporation of [3H]uridine into total and poly A+ RNA. Also, the hypotonic treatment did not alter specific Sertoli cell functions, i. e., secretion of Sertoli cell factor (inhibin) and stimulation of cAMP levels by follicle stimulating hormone in 2-d cultures. Androgen receptor concentration per dish was also not changed. Changes in several general metabolic parameters observed after hypotonic treatment of 2-d cultures were attributed primarily to loss of contaminating germ cells. Consequently, hypotonic treatment can be used to eliminate contaminating germ cells from the Sertoli cell cultures without apparent detrimental effects on a number of Sertoli cell biochemical parameters. This may be of considerable importance when the purity of Sertoli cells is critical for the interpretation of experimental data.

Studies of human granulocyte phagocytosis stimulation by tuftsin.

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMNs), is now known to bind to both PMNs and monocyte-macrophages, affecting their phagocytosis and other functions. The potential roles of tuftsin in surgery-related infections have been documented using animal models. However, there have been some difficulties in demonstrating the phagocytosis-stimulating activity of tuftsin. In view of this, we have developed a suitable human PMN phagocytosis assay for tuftsin and performed preliminary kinetic studies. The assay was performed on 24-well plates between PMNs and fluorescent microspheres. The greatest effect of tuftsin over the control was observed under the following conditions: 15 min incubation at 37 degrees C with 5 micrograms/ml tuftsin and a 50:1 ratio of particle to PMN. Particles bound on the surface of PMNs were removed by washing and trypsin treatment, followed by centrifugation through fetal bovine serum. This allowed us to utilize flow cytometry in this study. A flow cytometric procedure was then successfully adapted to human PMN phagocytosis that established a high correlation between microscopic evaluation and flow cytometry of phagocytosis. In addition to the above determinations of the percentage of phagocytic cells, we evaluated the effect of tuftsin on the number of particles engulfed by PMNs. Under the above optimum conditions, tuftsin has greater impact on the number of particles engulfed than on the percentage of phagocytic cells.

Tuftsin-enhanced thymidine incorporation by murine splenic monocytes.

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMN), is now known to bind to both PMN and monocyte-macrophages, affecting many of their functions. Administration of tuftsin induces leukocytosis in vivo. We have recently observed that while tuftsin remains in the cytoplasm upon binding and internalization in human PMNs, it translocates into the human monocyte nucleus, suggesting that tuftsin may directly affect growth of monocytes. We have therefore examined the effect of tuftsin on [3H]thymidine incorporation in fractions of murine splenocytes to identify a cell population responding to tuftsin. Tuftsin showed the greatest effect in [3H]thymidine incorporation of splenocytes over controls at optimum conditions of 2% fetal bovine serum and 1 microgram/ml of tuftsin. Splenocyte fractionation by Lymphocyte Separation Medium indicated that tuftsin primarily affects the mononuclear cell fraction; further fractionation revealed that tuftsin affects mostly the monocytes that adhered to plastic. We subsequently further purified the splenic monocytes by repeated plastic adhesion and Percoll gradient separation, to show that tuftsin increases [3H]thymidine incorporation of these highly purified monocytes.

Specific translocation of tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide, into the nuclei of human monocytes.

To delineate the mechanism of growth and differentiation activities of tuftsin (Thr-Lys-Pro-Arg), we examined the translocation of tuftsin after internalization by the target cells. We found using two independent techniques, fluorescence microscopy and autoradiography, that while in human polymorphonuclear leukocytes (terminally differentiated cells) the peptide remains in the cytoplasmic compartment, in monocytes it translocates to the nucleus. The ability of tuftsin to directly interact with DNA was documented by a large increase in the melting point of bovine DNA in the presence of tuftsin. It is suggested that the translocation, processing and action of tuftsin may depend on the differentiation state and/or on the type of effector cells. Also, tuftsin has the capacity to interact directly with DNA and, therefore, may have a potential for affecting gene activity.

Specificity and nature of the rapid steroid-stimulated increase in Sertoli cell nuclear RNA polymerase activity.

This report explores the ability of various steroids to rapidly stimulate Sertoli cell RNA polymerase II activity and to compete with [3H]-androgens for nuclear and cytosol binding sites. Nuclear RNA polymerase II activity was significantly stimulated by a 1 nM concentration of the androgenic compounds testosterone, dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one). R1881 (methyltrienolone) and 5 alpha, 17 beta-diol and also by the potent progestins 6 alpha methylprogesterone and R5020 (17,21-dimethyl-19-nor-4-pregna-3,20-dione). Progesterone, 17 alpha-hydroxyprogesterone, estradiol, androsterone, and 5 alpha-androstan-3 beta, 17 beta-diol were ineffective at 1 nM. Cytosol binding and nuclear accumulation of [3H]-androgen was effectively reduced by 100 fold molar excess of those androgens and progestins which stimulated RNA polymerase II activity. These data suggest that androgens and progestins bind to at least some of the same proteins in the Sertoli cell and may elicit the rapid stimulation of RNA polymerase II activity via a common mechanism. Agarose gel electrophoresis of the nuclear RNA synthesized as a result of exposure to testosterone indicated that is was heterodisperse and in part polyadenylated. Electrophoresis of the poly A+-RNA demonstrated that testosterone administration increased the incorporation of [3H]-UTP into RNA that was larger than 28 S.