Late-onset and long-lasting neutropenias in the young: A new entity anticipating immune-dysregulation disorders.

This study identifies a new chronic form of immune neutropenia in the young with or without detectable indirect anti-neutrophil antibodies, characterized by mild/moderate neutropenia low risk of severe infection (14%), tendency to develop autoimmune phenomena over the course of the disease (cumulative incidence of 58.6% after 20 years of disease duration), leukopenia, progressive reduction of absolute lymphocyte count and a T- and B-cell profile similar to autoimmune disorders like Sjogren syndrome, rheumatoid arthritis, and systemic lupus erythematosus (increased HLADR+ and CD3 + TCRγδ cells, reduced T regulatory cells, increased double-negative B and a tendency to reduced B memory cells). In a minority of patients, P/LP variants related to primary immuno-regulatory disorders were found. This new form may fit the group of "Likely acquired neutropenia," a provisional category included in the recent International Guidelines on Diagnosis and Management of Neutropenia of EHA and EUNET INNOCHRON ACTION 18233. The early recognition of this form of neutropenia would help clinicians to delineate better specific monitoring plans, genetic counseling, and potentially targeted therapies.

Effects of sodium hyaluronate in children with recurrent upper respiratory tract infections: results of a randomised controlled study.

Hyaluronic acid is a major component of many extracellular matrices that plays a role in the regulation of vasomotor tone and mucous gland secretion, and in the modulation of the inflammatory process in upper and lower airways. This pilot study was aimed at evaluating the effects of nasal washes with 9 mg nebulised sodium hyaluronate given for 15 days per month over 3 months in 75 paediatric patients with recurrent upper respiratory tract infections (URTI). Eligible patients were randomized to treatment with nasal washes containing 9 mg sodium hyaluronate plus saline solution or saline alone, according to an open-label, parallel group design, with blind observer assessment. Ciliary motility, which was assessed based on a 0-3 point rating scale (0 = absent, 1 = less than 5 minutes, 2 = greater than or equal to 5 and ≤ 10 minutes, 3 = greater than 10 minutes) was the primary study endpoint. The secondary efficacy variables included cytological (presence of neutrophils, eosinophils and mast cells), microbiological (presence of bacteria and mycetes), endoscopical (presence of adenoid hypertrophy and biofilm) and clinical (presence of rhinitis, post-nasal drip, nasal dyspnoea) parameters. The two treatment groups (mean age 7.5 years, 53percent of males) were comparable for baseline data, except a higher mean age in the control group than in the treated group. Treatment with 9 mg sodium hyaluronate was associated with significantly greater improvements (p less than 0.001 between groups) in primary outcome ciliary motility [odds ratio (OR) 13.61; 95 percent CI 4.51-41.00 in the univariate regression analysis that examined the probability of improvement]. Treatment with 9 mg sodium hyaluronate was also significantly superior to saline alone in adenoid hypertrophy (p less than 0.001; OR 14.72; 95 percent CI 4.74-45.68), presence of bacteria (p = 0.026; OR 2.95; 95 percent CI 1.15-7.55), neutrophils (p = 0.002; OR 4.51; 95 percent CI 1.75-11.62), rhinitis (p = 0.040; OR 10.47; 95 percent CI 3.10-35.31), nasal dyspnoea (p = 0.047; OR 3.80; 95 percent CI 1.09-13.19) and biofilm (p = 0.049; OR 9.90; 95 percent CI 2.61-37.47). Advantages of 9 mg of sodium hyaluronate over control on post-nasal drip and presence of mycetes (although evident) did not reach the level of statistical significance. The superiority of the treated group over saline alone was confirmed in a multivariate logistic regression analysis that took into account age as confounding factor. The number of days of absence from school was significantly lower in the 9 mg sodium hyaluronate group compared to controls (p less than 0.001 between groups). A 3-month intermittent treatment with 9 mg sodium hyaluronate with nasal washes plus saline solution was associated with significant improvements in ciliary motility and in cytological, microbiological, endoscopic and clinical outcomes compared to saline, in children with recurrent URTI.

Hyaluronan plus saline nasal washes in the treatment of rhino-sinusal symptoms in patients undergoing functional endoscopic sinus surgery for rhino-sinusal remodeling.

Hyaluronic acid is a major component of many extracellular matrices and plays a central role in the homeostasis of physiology in upper and lower airways. When topically administered following endoscopic sinus surgery, hyaluronic acid may be effective in functional recovery and in the prevention of recurrence of chronic rhinosinusistis. This pilot study was aimed at evaluating the effects of nebulised 9 mg of sodium hyaluronate given for 15 days per months over 3 months in 46 patients aged greater than 4 years who underwent functional endoscopic sinus surgery (FESS) for rhino-sinusal remodelling. Eligible patients were randomized to receive nebulised 9 mg sodium hyaluronate nasal washes plus saline solution or 5 ml saline alone (23 patients in each group), according to an open-label, parallel group design, with blind observer assessment. Treatment was administered by means of a nasal ampoule that allows nebulisation of particles with a median aerodynamic diameter greater than 10 micron, i.e. suitable for upper respiratory airways deposition. The efficacy variables included clinical (presence of nasal dyspnoea), endoscopical (ostium of paranasal sinuses, oedema, respiratory patency, synechiae, and appearance of nasal mucosa) and cytological (ciliary motility and presence of neutrophils, eosinophils, mast cells, bacteria, mycetes and bio film) measures. At the end of the study, patients expressed an opinion on the overall tolerability of treatment. The two treatment groups were comparable at baseline. Treatment with 9 mg of sodium hyaluronate was associated with significantly greater improvements compared to controls in nasal dyspnoea (p less than 0.001), presence of mycetes (p = 0.044), ciliary motility (p less than 0.001) and abnormalities in nasal secretions. A univariate logistic model, in which the odd ratio (OR) indicates the probability of success in the 9 mg sodium hyaluronate group compared to the control group, showed that the highest OR was observed for presence of nasal dyspnoea (OR = 21.36; 95 percent CI: 1.07 to 426.56), normal mucosa at endoscopy (OR: 9.62; 95 percent CI: 1.82 to 50.89), ciliary motility (OR: 7.27; 95 percent CI: 1.68 to 31.42) and presence of bio film (OR: 4.41; 95 percent CI: 1.26 to 15.40). Treatment with 9 mg sodium hyaluronate plus saline was well tolerated. A 3-month intermittent treatment with 9 mg sodium hyaluronate plus saline solution nasal washes following FESS for rhino-sinusal remodelling was associated with significant improvements in nasal dyspnoea, appearance of nasal mucosa at endoscopy and ciliary motility compared to saline alone.

Recurrent acute rhinosinusitis: a single blind clinical study of N-acetylcysteine vs ambroxol associated to corticosteroid therapy.

The aim of rhinosinusitis treatment is to restore sinusal eutrophism and to normalize ventilation and mucociliary transport. Frequently the improvement of sinusal physiological conditions is associated with a reduction of infections and pulmonary symptoms. The treatment of these diseases often requires the combination of medical and surgical strategies. In particular, the aim of the medical therapy is multiple: to treat the infection (with antibiotics), to reduce the mucosal swelling (with corticosteroids) and to improve mucus drainage (with mucolytics or muco-regulators). The use of atomized nasal douche, as a washing of the nasal fossas, is chosen because of its local action minimizing systemic adverse effects. The surgical treatment is secondary to medical failure, and it is focused on clearing the sinusal ostia in the sphenoethmoidal recess and the osteomeatal complex. In case of recurrent sinonasal diseases the importance of the surgical operation is represented by the fact that the medical treatment better reaches the target in the sinusal space. This study is focused on the primary medical treatment of acute recurrent rhinosinusitis. The patients who immediately needed surgical treatment were excluded from the study (because of the presence of an anatomical obstruction of the osteomeatal complex and/or the sphenoethmoidal recess, hence non-susceptible to improvement by medical therapy alone), and these patients were immediately addressed to undergo a CT scan examination in order to be involved in a future surgical programme. The medical treatment for those forms which do not require antibiotics (i.e. when infections are not involved), is based on the use of topical corticosteroids. While there are controversies on the real efficacy of adding mucolytic agents to the steroids, they are commonly prescribed in clinical practice, with the rationale of reducing viscosity and improving clearance of mucus in order to help the restoration of the physiological sinus conditions. The primary aim of the medical treatment is to reduce the number of acute episodes and thus to increase the time between the exacerbations, allowing a good quality of life without necessitating surgical procedure.

Dreaming experience as a useful diagnostic clue for syncopal episodes.

BACKGROUND: The differential diagnosis between epileptic seizures and syncopes is a common occurrence in clinical practice. The manifestations of seizure and syncope sometimes overlap, and available diagnostic testing often not provides a conclusive answer. Syncope is often preceded by a symptom complex characterized by lightheadedness, generalized muscle weakness, giddiness, visual blurring, tinnitus, and gastrointestinal symptoms. These subjective symptoms are very important in guiding the diagnosis. In our experience, the impression of coming out of a dream after the syncopal episode is a subjective symptom commonly reported by patients, if questioned. METHODS: To verify the occurrence of dreaming experience after syncope and after generalized tonic-clonic seizures (GTCS) and its diagnostic value in differential diagnosis, we asked 100 patients with GTCS and diagnosis of idiopathic generalized epilepsy (Group 1) and 100 patients with a certain diagnosis of syncope (Group 2) whether they have never felt the impression of coming out of a dream after the loss of consciousness (GTCS or syncope, respectively). RESULTS: In Group 1, nobody referred the dreaming experience, whereas in the syncope group, 19% of patients referred this subjective symptom. CONCLUSIONS: Dreaming experience seems to be an additional useful diagnostic clue for syncopal episodes, helping the clinician to differentiate them from seizures.

Aerosol therapy with thiamphenicol glycinate: a retrospective study on efficacy and safety in a group of sixty-six oncological patients.

The purpose of this paper is to present the effectiveness of aerosol administration of TG in a group of oncological patients. Thiamphenicol is an antimicrobial agent active in the treatment of infection of different etiology and localisation due to its broad spectrum of antimicrobial activity as well as its pharmacokinetic properties. The data of the retrospective study analysis of the activity of TG, administered to oncological patients affected by infections of the respiratory tract, showed that TG administered alone or in association with other antibiotics was globally effective in more than 95% of patients. These positive results were obtained in immunologically compromised patients. The therapeutic advantages of using TG are: ease of use - aerosol therapy permits good local action; tolerability - no adverse reaction or intolerance; the possibility of using it in an ideal association with other antibiotics to combat the infectious pathology.

Serum/glucocorticoid-induced protein kinase-1 facilitates androgen receptor-dependent cell survival.

Androgen receptor (AR) is a critical factor in the development and progression of prostate cancer. We and others recently demonstrated that eliminating AR expression leads to apoptotic cell death in AR-positive prostate cancer cells. To understand the mechanisms of AR-dependent survival, we performed a genome-wide search for AR-regulated survival genes. We found that serum/glucocorticoid-induced protein kinase-1 (SGK-1) mRNA levels were significantly upregulated after androgen stimulation, which was confirmed to be AR dependent. Promoter analysis revealed that the AR interacted with the proximal and distal regions of the sgk1 promoter, leading to sgk-1 promoter activation after androgen stimulation. Functional assays demonstrated that SGK-1 was indispensable for the protective effect of androgens on cell death induced by serum starvation. SGK-1 overexpression not only rescued cells from AR small-interfering RNA (siRNA)-induced apoptosis, but also enhanced AR transactivation, even in the absence of androgen. Additionally, SGK-1 siRNA reduced AR transactivation, indicating a positive feedback effect of SGK-1 expression on AR-mediated gene expression and cellular survival. Taken together, our data suggest that SGK-1 is an androgen-regulated gene that plays a pivotal role in AR-dependent survival and gene expression.

From modeling to remodeling of upper airways: Centrality of hyaluronan (hyaluronic acid).

After traumatic events (accidental or surgical), the respiratory tract activates specific and prolix repairing mechanisms which tend to claw back the primitive differentiated state. The attempt of reactivation of the normal tissue functions is called 'remodeling' and its aim is to reinstate the modeling mechanisms that existed before the damaging event or the pathology's establishment. Endoscopic sinus surgery represents the gold standard treatment for inflammatory, malformative, benign, and, in selected cases, malignant diseases. The surgical technique is commonly described as minimally invasive as the nostrils are used as an access route and therefore does not leave any external scars. Currently, the surgical procedures, even though minimally invasive regarding the way in, are in fact widely destructive towards the surgical target. The healing process and re-epithelialization will depend on the amount of bony tissue that has been exposed and it will be important to stratify the different surgical typologies in order to foresee the increasing difficulty of mucosal healing process. As far as upper inflammatory diseases are concerned, recent studies demonstrated how intranasal hyaluronic acid can positively regulate mucosal glands secretion and modulate inflammatory response, being a useful tool for the improvement of remodeling after endoscopic sinus surgery. Acid has shown to be able to regulate mucosal glands secretion and modulate the inflammatory response.

Steroidogenesis in experimentally induced atretic follicles of the hamster: a shift from estradiol to progesterone synthesis.

The effects of phenobarbital-delayed ovulation on in vitro steroidogenesis and aromatase activity of LH-stimulated preovulatory follicles was ascertained in the cyclic hamster. After 3 days of ovulatory delay, antral follicles exhibited early signs of atresia (degenerating granulosa cells and pyknotic nuclei within oocytes). On days 2 and 3 of delay, LH-stimulated follicles produced more progesterone (P 15--20 ng P/follicle . 90 min) in vitro than control proestrous follicles (2.5 ng P/follicle . 90 min). Concomitant with the increase in LH-stimulated P production was a decline in the in vitro production of androstenedione (A) and estradiol (E2) on day 2 (A, 5 ng/follicle . 90 min; E2, 4 ng) and day 3 (1 ng A, less than 0.2 ng E2)-proestrous controls: 8 ng A or E2/follicle . 90 min. The steroidogenic profile of 1-day delayed follicles was similar to proestrous controls. A similar E2-P shift was observed in vitro when the steroidogenic profile of preovulatory follicles on the morning of proestrus (2.5 ng P, 8 ng A or E2) was compared with post-LH surge follicles (25 ng P, less than 1 ng A or E2) and with new corpora lutea (36 ng P, less than 0.2 ng A, less than 1 pg E2). A slight increase in the aromatizing activity of follicles was observed on days 1 and 2 of delay. However, atretic antral follicles (day 3 of delay) exhibited an aromatizing capacity similar to that of proestrous controls. Collectively, these results indicate that the loss of E2-synthesizing capacity in atretic antral follicles (induced by ovulatory delay) is due to the lack of A precursor. In addition, the results indicate that phenobarbital-delayed ovulation induces a shift from E2 to P synthesis in 3-day delayed follicles (early atretic). This E2-P shift is similar to that in Graafian follicles on the afternoon of proestrus but occurs at a much slower rate.

Tumor necrosis factor alpha alters follicular steroidogenesis in vitro.

Preovulatory follicles from cyclic proestrous rats were incubated in vitro in M199 for up to 24 hours with various doses of human tumor necrosis factor alpha (TNF). Stepwise increases in progesterone (P) production (20-80 ng/ml/2 follicle/24 hours) were observed with 30-300 pM TNF; 3000 pM TNF reduced (p less than 0.001) P production compared to 300 pM TNF but it was still higher (p less than 0.001) than controls. Follicular androstenedione production was inhibited by low doses of TNF (30-60 pM TNF) and stimulated by 3000 pM TNF. TNF did not alter follicular estradiol (E2) production. The time course studies using 300 pM TNF revealed that follicular P production did not increase significantly until 24 hours in culture unlike LH (160 pM) which increased P by 6 hours. Preincubation of 30 pM TNF with a monoclonal antibody to human recombinant TNF alpha prevented the increase in follicular P production observed at 24 hours in TNF-treated controls. These results indicate that in vitro TNF stimulates follicular P production but only after a lag period and thus provide a basis for future studies elucidating the role of TNF in follicular development.

Involvement of protein kinase C in regulating tumor necrosis factor alpha-stimulated progesterone production in rat preovulatory follicles in vitro.

Tumor necrosis factor alpha (TNF) increased progesterone production in preovulatory rat follicles in vitro. More than 1 h in the presence of TNF was needed to enhance progesterone secretion, which was only seen after 24 h of culture. Neither cAMP nor cGMP levels in media and follicles increased either at short (5-20 min) or long periods (6-24 h) after TNF stimulation. The protein kinase C (PKC) inhibitor, H-7, blocked TNF-stimulated progesterone in a dose-dependent manner (1-300 mM), with 50% inhibition corresponding to 5.2 microM H-7, it also blocked LH-stimulated progesterone production, but higher doses were needed (50% inhibition corresponding to 54.5 microM H-7). However, the cAMP- and cGMP-dependent protein kinase inhibitor, HA1004, did not block TNF stimulated progesterone. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased progesterone maximally at 32 nM and above. Low doses of PMA in combination with TNF increased progesterone levels above that stimulated by PMA alone; however with the highest does of PMA (320 nM), TNF was unable to increase follicular progesterone secretion. The time course of progesterone stimulation by PMA was similar to that of TNF. H-7 also blocked PMA and PMA + TNF stimulated progesterone accumulation, with a 50% inhibition corresponding to 4.2 and 4.1 microM H-7, respectively. These results indicate that PKC may be a mediator of TNF-stimulated progesterone secretion in preovulatory rat follicles.

Lipopolysaccharide inhibits rat ovarian thecal-interstitial cell steroid secretion in vitro.

Lipopolysaccharide (LPS), a major component of gram-negative cell walls, is a potent immunostimulator. Treatment of monocytes/macrophages in vitro with LPS induces the secretion of cytokines such as tumor necrosis factor-alpha and interleukin-1 alpha, -1 beta, and -6. LPS is thought to require LPS-binding protein or CD14 to act at low concentrations (< 100 ng LPS/ml). In the present study, rat ovarian thecal-interstitial cells (TIC) were cultured in a serum-free culture system (in the absence of LPS-binding protein or soluble CD14) and challenged with LPS. Treatment with LPS led to a dose-dependent (1-100 ng LPS/ml) decrease in LH-stimulated progesterone and androstenedione secretion. LPS had no effect on radiolabeled hCG binding to TIC homogenates or cAMP accumulation in culture medium. LPS treatment was associated with an increase in interleukin-6 bioactivity in the medium of thecal-interstitial cell cultures; however, tumor necrosis factor-alpha bioactivity was undetectable. Herbimycin A, an src tyrosine kinase inhibitor, blocked the actions of LPS and was associated with an increase in cAMP accumulation in TIC culture medium. The results suggest that LPS can act directly on ovarian thecal-interstitial cells and that this can occur in a LPS-binding protein/CD14-independent manner. The actions of LPS appear to be specific and require a nonreceptor tyrosine kinase.

Effects of tumor necrosis factor-alpha in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target.

Tumor necrosis factor-alpha (TNF), a pleiotropic cytokine localized within the ovary, alters follicular steroidogenesis. Preovulatory follicles dissected from ovaries of normal cyclic adult rats on the morning of proestrus exhibit steroidogenic and histological signs of atresia after 24 h of culture under the conditions of 5% CO2 and air. Follicles cultured for 24 h in 5% CO2 and 95% O2 appeared histologically and steroidogenically healthy. Under both culture conditions, human recombinant TNF (5 ng/ml) significantly increased the production of pregnenolone, progesterone, 20 alpha-dihydroprogesterone, and 17 alpha-hydroxyprogesterone by the follicles. Follicles cultured in 5% CO2 and air exhibited no change in androstenedione or estradiol production compared to control follicles incubated without TNF. In contrast, follicles cultured in 5% CO2 and 95% O2 responded to TNF with increased androstenedione and estradiol production. Separation of the thecal and granulosa compartments indicated that the increased progestin production observed in the whole follicle in response to TNF originated from the theca. TNF significantly inhibited basal and FSH-stimulated progesterone production from the granulosa of preovulatory follicles. Exogenous substrate added to whole follicles cultured in the presence or absence of TNF indicated that TNF enhanced the conversion of 25-hydroxycholesterol to pregnenolone. These studies reveal that TNF enhanced steroidogenesis in both healthy and atretic follicles and that this action of TNF is on the theca, where TNF increases the conversion of cholesterol to pregnenolone. The data imply that TNF has differential effects on thecal and granulosa steroidogenesis.

Acute effects of exogenous progesterone on follicular steroidogenesis in the cyclic rat.

The effects of exogenous progesterone (P) on follicular steroidogenesis and serum gonadotropins in the cyclic rat were determined using in vivo and in vitro approaches. A single sc injection of 800 micrograms progesterone at 0900 h on diestrus II (day 0) consistently postponed ovulation by 24 h in 4-day cyclic rats, with no change in the ovulation rate. The preovulatory surges in serum LH and FSH were delayed by 1 day and occurred on day 2 in the P-treated animals. After the injection of 800 micrograms P at 0900 h on diestrus II, peak serum levels of P were reached 3 h later and thereafter decreased to basal levels by 1500 h on day 1. Within 1 h after injecting P, serum LH and FSH decreased to values below control levels; serum LH did not recover during the next 48 h, whereas serum FSH returned to basal levels within 6 h. An abrupt fall in serum 17 beta-estradiol occurred within 1 h after the administration of P. This drastic decline in serum estradiol was paralleled by temporally related decreases in serum LH and FSH but not by changes in serum testosterone. By 0900 h on day 1 (24 h after P injection), serum and estradiol returned to values within the range observed on diestrus II, then gradually increased to proestrous values by day 2. Within 3 h after the administration of P, the initial content and the in vitro production rate of P, testosterone, and 17 beta-estradiol by antral follicles decreased in the P-treated animals compared to that in follicles of control animals, whereas the follicular content the production rate of 20 alpha-dihydroprogesterone were unaffected. Thus, in the cyclic rat, the inhibitory effects of exogenous P on follicular steroidogenesis are mediated in this dose range through the hypothalamic-pituitary axis, especially via lowering serum LH.

Src tyrosine kinase activity is related to luteinizing hormone responsiveness: genetic manipulations using mouse MA10 Leydig cells.

The Src family of tyrosine kinases play an important role in various signal transduction pathways in many different cell types, however, the role of these kinases in steroidogenic cells has not been examined. In the present study, genetic approaches were used to directly alter Src tyrosine kinase activity in mouse MA10 Leydig cells in order to determine the effect of changes of Src activity on LH-responsiveness with regard to cAMP and progesterone secretion. MA10 cells expressing a dominant negative Src (MA10(Srck-3)) secreted more cAMP and progesterone in response to LH than control transfected cells. Phosphodiesterase activity was decreased in MA10(Srck-3) cells. Conversely, MA10 cells expressing a temperature sensitive Src (MA10(tsUP)) lost LH-responsiveness with regard to cAMP and progesterone secretion at the Src active temperature (35 degrees C). It is concluded that Src tyrosine kinase has an important role in regulating steroid secretion in MA10 Leydig cells. This regulation may in part be due to Src modulation of phosphodiesterase activity, although other components of the LH-signaling pathway may be involved.

Cyclic Djungarian hamsters, Phodopus campbelli, lack the progesterone surge normally associated with ovulation and behavioral receptivity.

Serum levels of 17 beta-estradiol and progesterone were assayed at 4- to 5-h intervals across the estrous cycle in female Djungarian hamsters, Phodopus campbelli. The pattern of secretion for estradiol is similar to that described for the rat and the Syrian hamster, rising slowly from a baseline of 54 +/- 2 pg/ml during the morning of estrus to a peak of 187 +/- 16 pg/ml in the early afternoon of proestrus, then falling rapidly back to baseline levels. The pattern of progesterone secretion is significantly different from that of any estrous cycle previously described for a 4-day cyclic mammal. There is no evidence for a significant peak in serum progesterone levels associated with ovulation and receptivity. The highest levels of serum progesterone occur during the early afternoon of diestrous day 2 (8046 +/- 1063 pg/ml). The lowest levels of serum progesterone occur in the early morning of proestrus (720 +/- 125 pg/ml). During the period of sexual receptivity only 2504 +/- 654 pg/ml progesterone are found in the serum. Ovariectomized females show behavioral receptivity after 48 h of treatment with 50 micrograms estradiol benzoate/kg BW in sesame oil. Progesterone is not necessary for receptivity and will not facilitate receptivity when administered in conjunction with a subthreshold dose of estrogen. The presence of tubular ova confirms the time of ovulation to be a period of approximately 7 h between 2200 h on proestrus and 0500 h on estrus. Histological examination of ovarian sections from each of the 4 days of the estrous cycle shows follicular development to differ from that in the Syrian hamster in that the total number of follicles in the ovary is variable and low, and the ovulated follicles (new corpora lutea) are blood filled. Djungarian hamster primary follicles averaged 83 +/- 2 microns in diameter, and preovulatory follicles averaged 455 +/- 17 microns in diameter. The total number of intact follicles per ovary rose from 21 +/- 5 on the evening of estrus to 73 +/- 29 on the evening of diestrous day 2, then fell to 32 +/- 26 by the evening of proestrus. Preovulatory follicles increased in number from 1.0 +/- 0.6/ovary early on estrus to a maximum of 4 +/- 1 on the evening of diestrous day 2. However, at ovulation a combined total of only 5.1 +/- 0.8 ova are shed.

Tumor necrosis factor-alpha induces clustering in ovarian theca-interstitial cells in vitro.

Tumor necrosis factor-alpha (TNF) has been implicated in the regulation of steroidogenesis in theca-interstitial cells (TIC). The purpose of this study was to evaluate any change in TIC morphology during the time course of TNF-induced inhibition of LH-stimulated androstenedione production. Ovaries from immature hypophysectomized rats were enzymatically digested and highly purified TIC were obtained by density gradient centrifugation. TIC treated with TNF (0.1-10 ng/ml) demonstrated distinct clustering in the presence and absence of LH (50 ng/ml). The number of clusters and the mean area per cluster were greatest after 4 days as a result of treatment with 1 or 10 ng TNF/ml. In addition, a dose-dependent inhibition of LH-supported androstenedione production was induced by TNF. TNF also inhibited LH-induced androstenedione in TIC after 2, 4, or 6 days of continuous LH treatment, and TIC clustering still occurred. TIC clustering was impeded by the protein kinase inhibitor H7 at 10 microM; however, the protein kinase inhibitor, HA 1004 (5 microM), did not inhibit TNF-induced clustering in TIC. Since H7 blocked TNF induced clustering, but did not block TNF inhibition of LH stimulated androstenedione synthesis, it is suggested that alternate signal transduction pathways for TNF induced inhibition of LH-stimulated androstenedione and stimulation of clustering of TIC may exist. The results also indicate that the TNF-induced TIC clustering may be independent of the TNF-induced inhibition of LH-stimulated androstenedione production and states of LH-induced differentiation of TIC.

Tumor necrosis factor-alpha attenuation of luteinizing hormone-stimulated androstenedione production by ovarian theca-interstitial cells: inhibition at loci within the adenosine 3',5'-monophosphate-dependent signaling pathway.

Tumor necrosis factor-alpha (TNF) blocks LH-stimulated androstenedione production by immature rat theca-interstitial cells (TIC) in vitro. The mechanism for TNF inhibition of LH-induced androstenedione is unknown and was investigated. LH stimulation of androstenedione synthesis in TIC is mediated via a cAMP-dependent signaling pathway. LH-stimulated cAMP in TIC-conditioned medium was reduced in a biphasic manner by TNF at 1 and 48 h, but not at 4 and 24 h. To determine whether inhibition of cAMP resulted from TNF interference of LH binding, TIC were given TNF for 24 and 48 h, and LH binding was determined. TNF inhibited LH binding at 24 and 48 h. Scatchard analysis revealed a TNF-induced decrease in LH receptor number without altered affinity. TIC were given TNF and cAMP analogs [N6-benzoyl-cAMP, 8-thiomethyl-cAMP, 8-(6-aminohexyl)amino-cAMP, and N6-2'-O-(Bu)2cAMP], which selectively activate cAMP-dependent protein kinase (PKA) type I and/or PKA type II, respectively. At 48 and 96 h, TNF blocked androstenedione production stimulated by all combinations of cAMP analogs; however, androstenedione synthesis recovered by 48 h after removal of TNF. Peak PKA activity in TIC was observed at 30 min in the presence of LH or cAMP analogs. LH- or cAMP analog-directed PKA activity was inhibited after concomitant exposure to TNF; however, a 24-h pretreatment with TNF did not affect cAMP analog-stimulated PKA activity. The results indicate that in the modulation of steroidogenesis, TNF acts at multiple sites in the PKA pathway. First, TNF suppresses LH-stimulated cAMP production by TIC. Secondly, inhibition of cAMP may result from TNF attenuation of LH binding, and thirdly, TNF inhibits PKA activity of TIC and, thus, attenuates androstenedione production.

Expression of tumor necrosis factor-alpha in the rat ovary.

Tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in rat ovaries was detected by Northern blot analysis and by reverse transcription-polymerase chain reaction (RT-PCR). Northern blot analysis of total cellular RNA (tcRNA) isolated from adult rat ovaries showed a single distinct band at approximately 2 kilobases, while rat white blood cells, used as control, also showed a second lower band at approximately 0.5 kilobases. TNF-alpha expression in ovaries was also detected by RT-PCR, using RNA-specific primers. Southern blot analysis of the RT-PCR products showed a single band of the expected 500 base pairs, from the ovary and white blood cells, using either tcRNA or polyA+ RNA. In order to validate the RT-PCR product, it was digested with restriction enzymes, HhaI, HindIII, and XhoI. The results indicate that the ovarian TNF-alpha mRNA does not contain major alterations with respect to the known structure of rat TNF-alpha mRNA. Ovarian RNA was also subjected to PCR without previous RT in order to amplify, if any, contaminating genomic DNA. A single band of approximately 900 base pairs was observed, which contained introns 1 and 2 (determined by restriction enzyme digestion). In summary, these results indicate that rat ovaries contain TNF-alpha mRNA which makes it a likely source of local TNF-alpha secretion. The possibility exists that ovarian cells and/or macrophages are the source of the ovarian TNF-alpha mRNA.