Effect of chronic treatment with [D-Ala6, des-Gly-NH2 10]LHRH ethylamide on ovarian histology in the rat

Summary The effect of chronic treatment with the LHRH agonist [D-Ala₆, des-Gly-NH₂ ¹⁰]LHRH ethylamide (LHRH-A) on ovarian histology was studied in adult female rats injected with 5 g of the peptide, once every second day, for 0, 2,4 or 8 weeks. Using light microscopy, we examined the number of small, medium, large and atretic follicles, as well as the number of normal and regressing corpora lutea. Using the point-counting method, we measured the relative surface occupied by luteal cells. It was found that treatment with LHRH-A for up to 8 weeks led to no significant change in any of the parameters studied. Moreover, resumed meiotic maturation of an abnormally high number of atypical fragmentations of the ova could not be observed. At least at the dose used, the LHRH agonist does not appear to induce any histological alteration in ovarian morphology and supports the potential clinical use of LHRH agonists as a new approach in femal contraception and for the treatment of estrogendependent pathologies.     

Effect of an LHRH Analog on Growth and Hormone Receptor Levels in DMBA-Induced Mammary Tumors in the Rat

Abstract

Dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat are well known to be hormono-dependent. Daily injections of an LHRH agonist, [D-Ala⁶, des-Gly-NH₂¹⁰]LHRH ethylamide (LHRH-A), 1 ug daily, for 38 days results in a 35% decrease in the number of tumors present at the beginning of the experiment compared to a decline of 45% after ovariectomy and of 8% in the control group. This is accompanied by a marked reduction in ovarian LH and FSH receptors. LHRH-A treatment also resulted in reduction in the number of progesterone and prolactin receptors in the tumors. In addition, an increase in plasma LH and FSH and a decline in plasma prolactin (PRL) concentrations are observed. The mechanisms by which the LHRH agonist induces its antitumoral effect probably relate to an ovarian desensitization to LH and FSH with a concomitant decrease in circulating levels of estrogen and prolactin, two well known stimuli for the growth of DMBA-induced mammary tumors.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Increased testicular 5α-androstane-3α, 17β-diol formation induced by treatment with [D-Ser(TBU)6, des-Gly-NH210]LHRH ethylamide in the rat

While the intact male adult rats respond to LH with a predominant increase of testicular and plasma testosterone levels, the response to LH stimulation in animals treated with the LHRH agoriist, [D-Ser(TBU)⁶, des-Gly-NH₂10]LHRH ethylamide is characterized by a major production of 5α-androstane-3α, 17β-diol. The marked increase of 5α-androstane-3α, 17β-diol levels in the presence of a 90% decrease of testosterone concentration strongly suggests that 5α-reductase and 3α-hydroxysteroid oxidoreductase activities are increased during testicular desensitization induced by treatment with the LHRH agonist.     

The ontogeny of components of the renin–angiotensin system in the porcine fetal ovary

There is an autonomous renin–angiotensin system (RAS) in the adult ovary. Renin is present in the primitive kidney, and the fetal ovary develops from the nephrogenic ridge. We hypothesised that components of the ovarian RAS would be present from early gestation, with potential roles in ovarian development. We studied fetal pig ovaries from approximately day 45 (～0.39 gestation) to term and measured mRNA (RT-PCR) for prorenin, angiotensinogen and the angiotensin II (AngII) Type 1 and 2 receptors (AT₁ and AT₂), and protein expression (Western blot) and localization (immunohistochemistry) of the AT₁ and AT₂ receptors. mRNA for prorenin was present in relatively low abundance from at least day 45 and rose to ～day 75 of gestation, whilst mRNA for angiotensinogen rose steadily. mRNA for the AT₁ receptor was present from approximately day 45 and did not alter significantly with increasing gestation but AT₂ receptor mRNA was initially high, falling sharply through pregnancy. The AT₁ receptor protein abundance fell steadily to term, whereas the AT₂ receptor protein did not change during gestation. Both receptors were localised in the surface epithelium and egg nests, the granulosa cells of primordial, primary and secondary follicles, and the oocytes of all except the secondary follicles. Collectively, our results support the hypothesis that there is a functional RAS in the fetal ovary from at least approximately day 45 of gestation until term and that it may have a paracrine role in ovarian growth and development.     

Testosterone treatment augments LHRH‐a induced ovulation vin greenback flounder

Greenback flounder were treated with either an injection of des Gly¹⁰ [D‐Ala⁶] LHRH ethylamide (LHRH‐a), a silastic pellet implant of testosterone (T) or LHRH‐a+T. LHRH‐a and LHRH‐a+T induced more ovulatory events than in control fish or fish treated with T alone. T treatment did not significantly increase the number of ovulatory events above control levels. Treatment with LHRH‐a+T stimulated more ovulatory events than treatment with LHRH‐a alone, suggesting T increases pituitary responsiveness to LHRH‐a. Plasma levels of 17β‐estradiol (E ₂) were significantly higher in fish treated with LHRH‐a and LHRH‐a+T than in control fish or fish treated with T. Plasma levels of 17,20β‐dihydroxy‐4‐pregnen‐3‐one were not elevated above control levels. Daily ovulations, elevated E ₂ and T levels and presumably elevated GtH‐II levels, persisted well after the expected clearance time of exogenous LHRH‐a. Positive steroid feedback may have contributed to the processes of repeated final oocyte maturation and ovulation.     

Antagonism by an LHRH agonist of the steroidogenic effects of exogenous human chorionic genadotrophin in the rhesus monkey

Fourteen regularly cycling female rhesus monkeys were observed daily for menstruation and bled from the saphenous vein at regular intervals throughout the study. Plasma samples were assayed by RIA for progesterone levels. The animals were divided into 3 subgroups. The first (n=5) received daily subcutaneous injections of 1000 IU hCG from the 18th to 36th day following onset of menstruation. The second (n=7) received the same hCG treatment and was also implanted subcutaneously from the 18th to 40th days with 1.2 mg [Des-gly¹⁰, DTrp⁶, ProNHEt⁹] LHRH contained in cholesterol matrix pellets. The third (n=2) was untreated. Intermenstrual interval was significantly extended by hCG treatment. The extension was partially overcome by the LHRH agonist. The hCG-induced elevation in plasma progesterone to peak values over 17ng/ml was blocked by the LHRH agonist to give mean values not significantly different from control luteal phase levels. Plasma estradiol levels were unaffected by hCG or LHRH agonist.     

Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using ¹²⁵I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu^t)⁶]LHRH(1–9) ethylamide and an antagonist DpGlu¹, DPhe², DTrp^3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K_a values for the agonist, LHRH and the antagonist were 2.1 × 10⁹M^?1, 0.92 × 10⁸M^?1 and 0.76 × 10⁹M^?1, respectively, and the binding capacity was 116 fmoles/10⁶ pituitary cells.     

Effects of estrogen and progesterone on LHRH release from a hypothalamic synaptosomal fraction of ovariectomized rats

Mitochondrial-synaptosomal fractions (P₂) from the basomedial hypothalamus of adult ovariectomized rats were employed to study the effects of estradiol benzoate (EB) and progesterone (P) on the release of luteinizing hormone-releasing hormone (LHRH). Treatment of ovariectomized rats with 5 or 50 g of EB significantly reduced the total LHRH released from P₂ under both control and K⁺-stimulated conditions. Furthermore, rats given 50 g EB demonstrated cyclic variations in the magnitude of inhibition of LHRH release. Comparison of LHRH release from P₂ of rats sacrificed at 0900 hr with that from those sacrificed at 1500 hr revealed a small persistent facilitation of LHRH release each afternoon. This facilitation, associated with an increase in the soluble component of LHRH release, was absent when rats also received 5 mg of P. No effects on LHRH release were observed when 17-estradiol alone or when P was applied to P₂ in vitro. The data show that the regulatory effects of estrogen and progesterone given in vivo on LHRH secretion can be observed in a subcellular fraction of the hypothalamus containing neurosecretory cell terminals.Supported by grants from the NIH, HD08389 and NS11753.U.S.P.H.S. Career Development Awardee, K04-HD00022     

Properties of LHRH release from a hypothalamic synaptosomal fraction of estrogen-primed ovariectomized rats

The release of luteinizing hormone-releasing hormone (LHRH) from mitochondrial-synaptosomal fractions (P₂) of basomedial hypothalamus was examined under various conditions. Less than 3% of the LHRH in P₂ suspensions was released under control conditions while the addition of 60 mM KCl or NaCl effected an 8-fold increase in LHRH as measured by radioimmunoassay. Equiosmolar sucrose effected only a 1.8-fold increase in LHRH release. The stimulatory effects of both Na⁺ and K⁺ were significantly inhibited by Mn²⁺ or La³⁺. Two forms of released LHRH were observed, one soluble and the other particulate. Soluble LHRH release was effected by hypertonic sucrose or 60 mM KCl and was not inhibited by Ca²⁺ antagonists. The release of particulate LHRH was unaffected by hypertonic sucrose, was stimulated 10-fold by 60 mM KCl, and was abolished with Ca²⁺ antagonists. These results suggest that the released soluble LHRH results from nonspecific leakage while the release of particulate LHRH reflects a Ca²⁺-dependent secretory process.     

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: A new tool for dissecting the molecular and cellular basis of LHRH physiology

Summary 1. Two LHRH neuronal cell lines were developed by targeted tumorigenesis of LHRH neuronsin vivo. These cell lines (GN and GT-1 cells) represent a homogeneous population of neurons. GT-1 cells have been further subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While considerable information is accumulating about GT-1 cells, very little is currently known about the characteristics and responses of GN cells.2. By both morphological and biochemical criteria, GT-1 cells are clearly neurons. All GT-1 cells immunostain for LHRH and the levels of prohormone, peptide intermediates, and LHRH in the cells and medium are relatively high.3. GT-1 cells biosynthesize, process, and secrete LHRH. Processing of pro-LHRH appears to be very similar to that reported for LHRH neuronsin vivo. At least four enzymes may be involved in processing the prohormone to LHRH.4. LHRH neurons are unique among the neurons of the central nervous system because they arise from the olfactory placode and grow back into the preoptic-anterior hypothalamic region of the brain. Once these neurons reach this location, they send their axons to the median eminence. With respect to the immortalized neurons, GN cells were arrested during their transit to the brain. In contrast, GT-1 cells were able to migrate to the preoptic-anterior hypothalamic region but were unable correctly to target their axons to the median eminence. These problems in migration and targeting appear to be due to expression of the simian virus T-antigen.5. While GT-1 cells are a homogeneous population of neurons, they are amenable to coculture with other types of cells. Coculture experiments currently under way should help not only to reveal some of the molecular and cellular cues that are important for neuronal migration and axonal targeting, but they should also highlight the nature of the cellular interactions which normally occurin situ.6. GT-1 cells spontaneously secrete LHRH in a pusatile manner. The interpulse interval for LHRH from these cells is almost identical to that reported for release of LH and LHRHin vivo. GT-1 cells are interconnected by both gap junctions and synapses. The coordination and synchronization of secretion from these cells could occur through these interconnections, by feedback from LHRH itself, and/or by several different compounds that are secreted by these cells. One such compound is nitric oxide.7. GT-1 cells have Na⁺, K⁺, Ca²⁺, and Cl^– channels. Polymerase chain reaction experiments coupled with Southern blotting and electrophysiological recordings reveal that GT-1 cells contain at least five types of Ca²⁺ channels. R-type Ca²⁺ channels appear to be the most common type of channel and this channel is activated by phorbol esters in the GT-1 cells.8. LHRH is secreted from GT-1 cells in response to norepinephrine, dopamine, histamine, GABA (GABA-A agonists), glutamate, nitric oxide, neuropeptide Y, endothelin, prostaglandin E₂, and activin A. Phorbol esters are very potent stimulators of LHRH secretion. Inhibition of LHRH release occurs in response to LHRH, GABA (GABA-B agonists), prolactin, and glucocorticoids.9. Compared to secretion studies, far fewer agents have been tested for their effects on gene expression. All of the agents which have been tested so far have been found either to repress LHRH gene expression or to have no effect. The agents which have been reported to repress LHRH steady-state mRNA levels include LHRH, prolactin, glucocorticoids, nitric oxide, and phorbol esters. While forskolin stimulates LHRH secretion, it does not appear to have any effect on LHRH mRNA levels.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

Demonstration and topographical distribution of LHRH receptors in the central nervous system in the normal and castrated male rat

The topographical distribution of [125I]-LHRH binding sites was studied on brain sections of adult male rat by quantitative autoradiography. High density of sites was observed in the hippocampus, amygdala and entorhinal cortex (4-7 fmol of LHRH bound/mg protein). Lower density of sites was observed in the septum and frontal cortex. The receptor density was not significantly modified at day 5 following castration. Under the same conditions the pituitary receptors were significantly increased. The presence of specific LHRH binding sites in the limbic system may explain the behavioural effect observed following intracerebroventricular injection of LHRH. However, their functions under physiological conditions remain to be elucidated.     

Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells

Leukemia inhibitory factor (LIF) is a glycoprotein that induces the differentiation of the monocytic leukemia cell line M1 but suppresses the differentiation of totipotent embryonic stem cells. In an attempt to define the normal cellular targets for LIF, the distribution of LIF receptors within hemopoietic and hepatic tissue was analyzed by binding cells with radioiodinated LIF (¹²⁵I-LIF) and subsequently carrying out autoradiography. Autoradiography demonstrated that in each he-mopoietic tissue examined cells of monocyte/macrophage lineage were the primary cell type labeled with ¹²⁵I-LIF. Moreover, both fetal and adult parenchy-mal hepatocytes displayed higher levels of labeling than either monocytes or macrophages. The number of receptors per positive cell varied from 150 for bone marrow monocytes to 2,000 for adult hepatocytes. In each case, however, binding was of high affinity, with an apparent K_D of 34–100 pM, and binding was specific, since labeling was competed for by unlabeled LIF but not a range of other structurally unrelated growth and differentiation factors. It is suggested that LIF may play a role in regulating macrophage function and hepatic acute phase protein synthesis in response to infection.     

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

Summary In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of ¹²⁵I-labelled human follicle-stimulating hormone (¹²⁵I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of ¹²⁵I-labelled human choriogonadotropin (¹²⁵I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.     

(Na,K)-ATPase activity of embryonic chick heart and skeletal muscles as a function of age

The specific activity of the membrane (Na⁺,K⁺)-ATPase of intact chick embryonic hearts (ventricles) was measured as a function of embryonic age. The membranes were prepared by a NaI extraction method of the 100 000 × g fraction which selectively removes much of the ouabain-insensitive Mg²⁺-ATPase. The specific activity of the myocardial (Na⁺, K⁺)-ATPase increased markedly during embryonic development from mean levels of about 3.0 μmoles P_i per h per mg protein at day 6 to 7.4 at day 16 and 11.0 at day 20 (1 day prior to hatching); the adult level was about the same as that of the 16-day-old chick. The relative activities with respect to that at day 16 (from paired experiments) averaged 43 % (day 6), 56 % (day 9), 73 % (day 13), 140 % (day 20), 115 % (day 23), 126 % (day 30) and 96 % (adult). There was a similar increase in relative activity of the (Na⁺,K⁺)-ATPase from chick skeletal (leg) muscles during development. If the total protein content per unit membrane area and the turnover number remain constant, the data indicate that the surface density of the (Na⁺,K⁺)-ATPase molecules increases during embryonic development; thus, the cation pumping capabilities of the cells should be enhanced if the surface area/volum ratio of the myocardial cells remains unchanged. However, the pumping capabilities of the very young cells must be sufficient to maintain the known high [K⁺]_i and low [Na⁺]_i already present; their internal activities actually change only to a small extent during development. Since there is a known increase in K⁺ permeability during embryonic development, thereby increasing the demand on the cation pump, the observed increase in activity of the (Na⁺,K⁺)-ATPase tends to compensate for this.     

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4⁺CD25⁺FOXP3⁺ circulating cells (CD4 T_reg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4⁺ T_reg cells rapidly decreased after primary tumour debulking, while CD8⁺CD25⁺FOXP3⁺ (CD8 T_reg) cells are not detectable in peripheral blood. Similar results on CD4 T_reg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T_reg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β₁ and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8⁺ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T_reg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.     

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct ³H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.