Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.

Radioimmunoassay of leukocyte (alpha) interferon and its application to some clinical conditions.

A radioimmunoassay for human leukocyte interferon alpha (alpha IFN) has been developed, using tracer produced by recombinant DNA (IFLrA) and polyclonal rabbit antiserum against partially purified leukocyte IFN (PP alpha IFN). Sensitivity was 4 units/ml using sequential saturation. Only lymphoblastoid IFN showed complete cross reactivity. Serum alpha IFN concentrations were measured in normal subjects and in patients with acute viral infections, bone and joint diseases, and malignancies. Some cases in the first two groups of patients had significantly elevated serum levels compared with controls. The pharmacokinetics of alpha IFN were studied in treated cancerous patients. Radioimmunoassay and biological assay gave similar and closely correlated results. Radioimmunoassay is thus a useful method for the routine assay of alpha IFN, especially in biological fluids containing low concentrations.

In vitro effects of thymopentin on the gamma-interferon production by peripheral blood mononuclear cells from normal subjects and from patients with rheumatoid arthritis.

The in vitro production of gamma-interferon (gamma-IFN) by peripheral blood mononuclear cells (MNC) was measured using a specific radioimmunoassay in 16 patients presenting with active rheumatoid arthritis (RA), in 14 patients with inactive disease, and in 36 control subjects (CS). Unstimulated cultures produced undetectable levels of gamma-IFN and did not incorporate tritiated thymidine. In response to phytohemagglutinin (PHA) 0.2 microgram/ml, MNC from active RA produced 9 times less, and under PHA 2.5 micrograms/ml, 4 times less gamma-IFN than did MNC from inactive RA or from CS. The uptake of tritiated thymidine was, however, similar in the 3 groups. In unstimulated cultures of the 3 groups, thymopentin (TP-5), at all concentrations tested, did not influence either the levels of gamma-IFN or the uptake of tritiated thymidine. In the presence of PHA 0.2 microgram/ml and TP-5, lambda-IFN levels were increased in CS, unchanged in inactive RA and reduced in active RA, whereas no changes were observed in the uptake of tritiated thymidine. Our results show that under our experimental conditions, TP-5 was able to increase the levels of gamma-IFN produced by normal MNC in vitro, but could not reverse the profound defect observed in active RA.

Effects of dexamethasone on the profile of cytokine secretion in human whole blood cell cultures.

EXPERIMENTAL OBJECTIVES: The interaction between the endocrine and immune systems is a very intriguing area. Endogenous glucocorticoids, as end-effectors of the hypothalamo-pituitary-adrenal axis, inhibit the immune and inflammatory responses and are used as immunosuppressive drugs in many inflammatory, autoimmune and allergic diseases. The aims of this study were to investigate the effects of dexamethasone on the profile of cytokine secretion in whole blood cell cultures from healthy subjects and to analyse the gender-related sensitivity to dexamethasone on each cytokine secretion. RESULTS: There was a significant inhibition by dexamethasone (from 1 to 100 nM) on the secretion of monokines (IL-1beta, IL-6, IL-8 and TNF alpha) and lymphokines (IL-2, IL-4, IL-10 and IFN gamma), either after LPS or PHA stimulation (P < 0.01). Interleukin 4 and IL-10 were less inhibited than IFN gamma (P < 0.05 at 1 nM, P < 0.01 at 10 nM and P < 0.001 from 100 nM to 10 microM). No gender difference was observed in the rate of inhibition of the secretion of each cytokine. CONCLUSION: This study shows that the inhibition of cytokine secretion by dexamethasone is more marked on Th1-type cytokines than on Th2-type cytokines. These data support the idea that glucocorticoids may induce a shift from the Th1 to Th2 profile of cytokine secretion.

Production of interferon gamma by peripheral blood mononuclear cells from normal subjects and from patients with rheumatoid arthritis.

A radioimmunoassay for human interferon gamma (IFN-gamma) has been carried out using a recombinant glycosylated interferon (Hu IFN-gamma) as tracer, the N.I.H. reference preparation (Gg 23-901-530) and a polyclonal rabbit antiserum. The assay is highly specific for IFN-gamma: there is no cross-reaction either with interferons alpha and beta, Interleukins 1 and 2, tumor necrosis factor alpha and beta or with various brain peptides. The sequential saturation procedure allowed a sensitivity of 0.4 U/ml with intra and between assay coefficients of variation less than 8 and 12%, respectively. The in-vitro production of IFN-gamma by peripheral blood mononuclear cells (P.B.M.C.) was also measured. In unstimulated cultures, IFN-gamma production remained undetectable, i.e. below the 0.4 U/ml sensitivity level. After stimulation of P.B.M.C. from normal subjects with increasing amounts of PHA, both the 3H-thymidine incorporation and IFN-gamma release followed bell-shaped curves. There was no significant difference of 3H-thymidine incorporation between PHA stimulated cultures (0.2 and 2.5 ug/ml) from normal subjects (36 cases) and those with active (16 cases) or non-active (14 cases) rheumatoid arthritis. At two PHA concentrations of 0.2 and 2.5 ug/ml, mononuclear cells from patients with active disease produced significantly less IFN-gamma than those from either controls or cases with non-active disease.

Radio-immunological study of the regulation of interleukin-2 production in peripheral blood mononuclear cell cultures from normal subjects and rheumatoid arthritis patients.

A radio-immunoassay for human T cell growth factor, also called Interleukin-2 (IL-2), has been carried out using a recombinant IL-2 preparation as tracer and a polyclonal rabbit antiserum. The assay is highly specific for IL-2: there is no cross-reaction with either type I and II interferons, epidermal growth factor or tumor necrosis factor alpha. Using the sequential saturation procedure the limit of sensitivity was 0.5 U/ml. Intra- and between-assay coefficients of variation were 8 and 11%, respectively. With this assay, IL-2 recovery in serum and peripheral blood mononuclear cell culture (P.B.M.C.) medium was 79 and 95%, respectively. In serum of 109 normal subjects and 102 rheumatoid arthritis patients mean IL-2 concentrations (+/- SD) were 1.5 +/- 0.5 U/ml and 1.4 +/- 0.4 U/ml respectively. The IL-2 production by P.B.M.C. in vitro was also studied. In unstimulated cultures, IL-2 release remained undetectable, i.e. below 0.5 U/ml. After stimulation of mononuclear cells from 36 normal subjects with increasing amounts of phytohemagglutinin (PHA), the 3H-thymidine incorporation followed a bell-shaped curve, the maximum response being observed at a 2.5 micrograms/ml PHA concentration. After a 72-hr mononuclear cell stimulation, IL-2 release increases with PHA concentrations ranging from 0 to 10 micrograms/ml. In patients with rheumatoid arthritis (R.A.), P.B.M.C. incorporated 3H-thymidine as in normal subjects. In contrast, mean +/- SEM IL-2 production by P.B.M.C. from patients with inactive RA (5 +/- 0.9) and active disease (1 +/- 0.5) was significantly lower than that from normal subjects (12 +/- 0.7 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

The value of a radioimmunological monitoring in cancer patients treated with interferon alpha.

Using a radioimmunoassay for human leukocyte interferon (IFN alpha), pharmacokinetic studies were carried out in twelve cancer patients given sequential intramuscular injections of Hu IFN alpha 2. Even though individual monitoring of serum IFN titers emphasized, for a given dose, marked quantitative variations of the observed maximum concentrations, their mean values were found to be dose-dependent (358 +/- 167 U/ml at 30.10(6) U and 1044 +/- 599 U/ml at 100.10(6) U doses). Comparison with bioassay results showed that IFN activities measured in sera were of the same order of magnitude as those calculated from radioimmunoassay standard curves. Data obtained from this series on observed peak time, half-life value and serum concentrations were consistent with those reported by the other groups using recombinant leukocyte interferon in clinical trial. Therefore, radioimmunoassay is an useful method for routinely assaying IFN alpha used either as antitumour or antivirus agent because of its high sensitivity (4 U/ml) and its simplicity.

[Tumor markers]. / Les marqueurs tumoraux.

Several substances, referred to as tumor markers, are associated with neoplasms development. The specificity of these cancer related substances or antigens depends on their nature (onco-fetal antigens, placental antigens) or on their concentration and physico-chemical forms (hormones, exocrine products, enzymes,...). On the basis of physico-chemical, immunochemical and biochemical analogies which exist between these tumor markers and substances normally found at particular times of life, a classification of these markers may be proposed. Tumor markers are almost constantly found within carcinoma cells by immunocytochemical techniques and are secreted by carcinoma explants in culture medium. On the hand, the release of tumor markers in biological fluids (blood, cerebrospinal fluid, urines,...) is less frequently detected by sensitive methods such as radioimmunoassay. Several factors are responsible for this discrepancy between the intra-tumoral presence of tumor markers and the lower incidence of their detection in biological fluids. These factors are discussed. These tumor markers have attracted considerable attention from pathologists and clinicians. Thus, detection of these substances, especially by immunocytochemical methods, may be related to a situation of neoplastic transformation and allow a functional classification superimposed to histological classification superimposed to histological classification of tumors. Moreover, ectopic production of hormones and/or neuromediators explains some clinical symptoms in cancer processes. Furthermore, products of normal cell activity at the origin of cancer (hormones, enzymes, exocrine products) when evidenced within the neoplastic cells or within serum might constitute a hormonal dependence index useful for therapeutical orientation. Finally, tumor marker levels are related to the local and systemic extension of the neoplasia and may be considered as valid index of prognosis. The determination of the levels of these tumor markers provides a quantitative criterion of the evolution of the neoplastic disorder and for following the efficacy or inefficacy of treatment.

[Dosage of prolactin in normal and pathological subjects (author's transl)]. / Dosage de la prolactine dans les conditions normales et pathologiques

Homologous radioimmunoassay of human prolactin was performed and used to investigate the secretion of this pituitary hormone under normal and pathological conditions. The following data were developed in this paper: --During the menstrual cycle, prolactin levels are more elevated during ovulatory and luteal phases than during follicular phase; --During pregnancy, prolactin levels progressively increase to reach their maximum at the end of gestation; --During lactation, prolactin levels increase for the two days following delivery, then progressively decrease; --Bromocryptine oral administration drastically reduces prolactin levels within the 24 hours as it inhibits the prolactin release normally induced by suckling. Assays of prolactin is useful to define the role of this hormone in the genesis of the galactorrhea. Bromocryptine as L Dopa reduce the hyperprolactinemia either functional or tumoral in origin. TRH provokes prolactin release which is more pronounced in females than in males. Pituitary prolactin reserve may be appreciated in several pathological conditions. It appears to be of first interest to assess the residual function of the pituitary gland after surgical removal of adenoma. Depuis 1971, la prolactine humaine peut être dosée dans les milieux biologiques par méthode radio-immunologique (G. BRYANT et coll., 1971; M. L'HERMITTE et coll., 1972; H. FRIESEN et coll., 1972; SINHA et coll., 1973). En 1974 nous avons proposé une méthode de dosage radio-immunologique homologue (A. REUTER et coll., 1976). Grâce à celle-ci nous avons exploré la sécrétion de cette hormone hypophysaire dans certaines conditions normales et pathologiques.

Effects of a hydrosoluble bacterial extract from Escherichia coli (OM-89) on cytokine production by peripheral blood mononuclear cells from healthy subjects and patients with rheumatoid arthritis.

OM-89 is a bacterial extract from escherichia coli, proposed as an immunomodulating drug for the treatment of rheumatoid arthritis (RA). Since immunological mechanisms may play a role in its action, the immunological effects of OM-89 were evaluated in vitro on peripheral blood mononuclear cells (PBMC) derived from healthy subjects and RA patients. Results indicated that in the absence of OM-89, production of the monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) is increased, while that of the lymphokines interleukin-2 (IL-2) and interferon-gamma (IFN-gamma is decreased by phytohemagglutinin (PHA)-stimulated PBMC from RA patients as compared with PBMC from healthy subjects. In the presence of PHA, OM-89 enhanced the production of IL-1 beta, TNF-alpha, IL-2, and IFN-gamma. IL-1 beta and IL-2 curves obtained using increasing amounts of OM-89 did not differ depending on the source of PBMC. By contrast, in the presence of increasing amounts of OM-89, TNF-alpha secretion significantly higher and IFN-gamma secretion significantly lower with PBMC from RA patients compared to PBMC from healthy subjects. These data indicate that OM-89 acts on monocytes and T cells directly and/or indirectly and suggest a possible clinical activity by OM-89 in RA relative to its immunological properties.

Immunomodulatory properties of cyclic hexapeptide oxytocin antagonists.

The thymic repertoire of neuroendocrine 'self' antigens has been previously described on the basis of the intrathymic expression of neurohypophysial (NHP)- and tachykinin-related peptide signals and receptors. According to that model, the cryptocrine signalling between thymic epithelial/nurse cells and thymocytes through NHP-related signals and receptors constitutes one accessory pathway in the process of T-cell differentiation and/or activation. A pharmacological manipulation of that novel type of cell-to-cell signalling was tested by the investigation of the immunomodulatory properties of novel cyclic hexapeptide oxytocin (OT) antagonists (MSD Research Laboratories). These compounds were found to significantly inhibit the productions of cytokines (mainly IL-1 beta and IL-6) elicited by anti-CD3 treatment of human whole blood cell cultures. Cytokine productions were more significantly reduced by OT antagonists in whole blood cell cultures derived from female volunteers than in those obtained from male donors, suggesting an influence of the gonadal steroid environment on the expression of NHP peptide receptors by immune cells. These observations support the concept of novel immunomodulating approaches through immune-specific neuropeptide antagonists, as well as the pharmacological value of such strategies in selective immunotherapy.

[Labeling of beta-endorphin and beta-lipotropin with iodine 125]. / Etude du marquage de la beta-endorphine (beta-END) et de la beta-lipotropine (beta-LPH) par l'iode 125.

5 micrograms of human beta-endorphin were labelled with 2 mCi 125I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 microCi/ug. Kept at + 4 degrees C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a 125I beta-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-beta-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 micrograms of human beta-lipotropin were labelled with 0.5 mCi 125I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 microCi/micrograms was obtained. It remained utilizable for 30 days when kept at + 4 degrees C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled beta-LPH was 32% for an anti-beta-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.

Serum concentrations of human chorionic gonadotrophin and its alpha and beta subunits. 1. During normal singleton and twin pregnancies.

Homologous radioimmunoassays have been developed for native hCG and its alpha and beta subunits. Their specificities were assessed by analysis of the inhibition ca and beta subunits. 1. During normal singleton and twin pregnancies.

Immunoreactive prolactin like material in the urine of women.

Immunoreactive prolactin-like material (Ir Prl) was detected in urines of eugonadal women during the luteal phase and in urines of pregnant and lactating women. The levels of Ir Prl urinary excretion per 24 h and of elimination per 100 ml of glomerular filtrate were highest in lactating women as compared to pregnant women; levels in pregnant women were elevated as compared to eugonadal subjects. Iv injection of thyrotropin releasing hormone (TRH, 200 micrograms) caused increased levels of urinary Ir Prl. The physiochemical characteristics of urinary Ir Prl of lactating women were investigated by filtrating urine samples on Ultragel Aca 54 in presence or absence of Trasylol. Urines, supplemented with Trasylol and analyzed either immediately or after storage at room temperature for 24 h, contained in addition to the 23,000 Mr monomeric form (25.2 +/- 7.4%), two fractions of high (greater than or equal to 70,000) or low (less than 23,000) molecular weight, respectively. The latter material largely predominated (73.5 +/- 7.3%). Urines kept at room temperature for 24 h without Trasylol showed variable but significant decreases in the monomer form with a parallel increase in high MW and fragmented forms. The extent of degradation of the monomer was directly proportional to the proteolytic activity of the urines as estimated by the Azocoll breakdown test. Contrary to what was observed with the urinary endogenous monomeric Prl, human pituitary Prl remained unaltered upon incubation in Trasylol-free urines up to 45 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diclofenac on plasma levels of immunoreactive prolactin, follicle stimulating hormone, luteinizing hormone, thyrotropin, and beta-endorphin in humans.

Prostaglandins have been shown to modulate the secretion of several pituitary hormones, suggesting that therapeutic doses of nonsteroidal anti-inflammatory drugs may change basal hormone levels. In this study, plasma levels of prolactin, follicle stimulating hormone, luteinizing hormone, thyrotropin and beta-endorphin were determined in 6 healthy men after administration of diclofenac, a prostaglandin synthesis inhibitor. The subjects were given 75 mg intramuscularly and 50 mg orally at 08.00 h the first day, 50 mg orally at 08.00, 12.00 and 20.00 h the second day and an additional 50 mg orally at 08.00 h the third day. Blood samples were collected throughout these 3 days. Diclofenac resulted in a significant and sustained decrease in plasma level of prolactin (p less than 0.005). The other hormones did not demonstrate significant change following diclofenac administration. These data suggest that administration of a prostaglandin synthesis inhibitor, such as diclofenac, selectively alters basal pituitary secretion of prolactin in humans without a detectable effect on plasma levels of other pituitary hormones. This study supports the hypothesis that prostaglandins are necessary for maintaining basal level of prolactin secretion in man.

Production of tumour necrosis factor-alpha, interferon-gamma and interleukin-2 by peripheral blood mononuclear cells of subjects suffering from rheumatoid arthritis.

Using radio-immunoassay methods, the production of tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) released by peripheral blood mononuclear cells (PBMC), maintained in culture and stimulated by phytohemagglutinin (PHA), was measured in normal subjects and patients with active or inactive rheumatoid arthritis (RA). Results indicated a dissociation between mitogenic response and secretion of mediators by PBMC under the influence of PHA in both normal controls and in patients with rheumatoid arthritis (RA). While [3H]thymidine incorporation was characterized by a rather bell-shaped curve with increasing concentrations of PHA, IL-2 and TNF-alpha displayed a linear dose-dependent increase. [3H]thymidine uptake by PBMC was in the same range in normal subjects as in patients with active and inactive RA, although cytokine secretion differed. The PBMC of patients with active RA produced less TNF-alpha, IL-2, and IFN-gamma than did those of the controls. In cases of inactive RA, the secretory response varied from subject to subject; mean values did not differ from those of normal subjects, except for those of IL-2 (p less than 0.01). The significance and the clinical relevance of these findings are discussed.

Prolactin levels during the menstrual cycle.

The levels of prolactin, FSH, LH, oestradiol and progesterone were measured daily during fourteen ovulatory cycles. The behaviour of FSH, LH, oestradiol and progesterone was classical. Non-systematic changes occurred in prolactin levels during the course of the menstrual cycle with the highest level being either during the ovulatory period or during the luteal phase. However, the mean level of prolactin was significantly higher during the ovulatory and luteal phases than during the follicular phase. A direct relationship between oestradiol and prolactin levels was noted, although there was no correlation between prolactin on the one hand and FSH, LH and progesterone on the other.

Serum concentration of human chorionic gonadotropin and its alpha and beta subunits. 2. Trophoblastic tumours.

Using specific homologous radioimmunoassays of native hCG and its alpha and beta subunits, we determined the levels of these glycoproteins in unfiltered maternal blood serially obtained in five non-invasive hydatidiform moles before and after evacuation. Some of these samples were assayed after gel filtration chromatography on Sephadex G 100. Twelve samples, obtained in cases of invasive trophoblastic tumour after ablative surgery and chemotherapy, were also assessed for their hCG, hCG alpha and hCG beta content. In unaborted moles, mean circulating levels of native hCG and free hCG beta were considerably increased (seven and thirteen times, respectively) as compared to normal pregnancies of the same age, whereas levels of free hCG alpha were either normal or slightly elevated. Chromatographic analyses of molar sera confirmed the presence of free circulating subunits, and separated hCG beta in its monomeric form from its higher molecular weight form, the latter being in greater quantity than in normal pregnancy sera. In contrast, the elution profile of serum native hCG was comparable in cases of normal and molar pregnancy. Successful curettage was accompanied by a return to normal levels of the native hCG and its alpha and beta subunits in 40-90 days. Persistence of tumour tissue was indicated by a slight increase in levels of native hCG and the beta subunit. Determination of alpha subunit level was less useful for the detection of any relapse.

Decreased corticosensitivity in quiescent Crohn's disease: an ex vivo study using whole blood cell cultures.

Corticosensitivity influences the degree and the duration of an inflammatory reaction by altering target cell responses to endogenous and/or exogenous glucocorticoids. Indeed, different clinical responses to glucocorticoids have been observed among patients with Crohn's disease, suggesting different degrees of corticosensitivity in these subjects. The purpose of this study was to compare the corticosensitivity of patients with quiescent Crohn's disease to that of healthy subjects (HS). Nineteen patients with quiescent Crohn's disease and 14 HS were studied; all patients were steroid-free for at least six months; 7 of the 19 were corticosteroid-dependent (CSD) and treated with nonglucocorticoid immunosuppressants at the time of the study. Corticosensitivity was measured by the inhibition of LPS-induced cytokine secretion in whole blood cell cultures treated with increasing concentrations (10(-9) to 10(-6) M) of dexamethasone. Tumor-necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta) were measured using specific immunoassays. Crohn's disease patients had a markedly decreased dexamethasone-mediated inhibition of TNF-alpha (P < 0.01), IL-6 (P < 0.001), and IL-1 beta (P < 0.01) compared to healthy subjects, with a shift of the dexamethasone dose-response curve to the right. No significant differences in the basal and LPS-stimulated secretion of the three cytokines were observed between CSD and non-CSD patients, and both subgroups of patients had similar degrees of dexamethasone-mediated cytokine inhibition. We conclude that patients with Crohn's disease have a significant decrease in the corticosensitivity of their leukocytes. This may be related to a specific genetic/constitutional background and/or could be acquired, due to inflammation-related endocrine and/or immune factors.