In vitro macrophage activity: Biphasic effect of prolactin and indirect evidence of dopaminergic modulation

Objective: Prolactin (PRL), a peptide hormone produced by the pituitary gland, is involved in the interaction between the neuroendocrine and immune system. Since dopamine receptor antagonists increase serum levels of PRL, both PRL and dopamine receptors might be involved in the modulation of macrophage activity, providing means of communication between the nervous and immune systems. This study evaluated the effects of PRL and the dopamine antagonist domperidone (DOMP) on macrophage activity of female rats. Methods: Oxidative burst and phagocytosis of peritoneal macrophages were evaluated by flow cytometry. Samples of peritoneal liquid from female rats were first incubated with PRL (10 and 100 nM) for different periods. The same procedure was repeated to evaluate the effects of DOMP (10 and 100 nM). Results: In vitro incubation of macrophages with 10 nM DOMP decreased oxidative burst, after 30 min, whereas the PMA-induced burst was decreased by DOMP 10 nM after 2 and 4 h. Treatment with PRL (10 and 100 nM) for 30 min decreased oxidative burst and rate of phagocytosis (10 nM). After 2 h of incubation, 10 nM PRL decreased oxidative burst and phagocytosis intensity, but increased the rate of phagocytosis. On the other hand, after 4 h, PRL 10 and 100 nM increased oxidative burst and the rate of phagocytosis, but decreased intensity of phagocytosis. Conclusions: These observations suggest that macrophage functions are regulated by an endogenous dopaminergic tone. Our data also suggest that both PRL and dopamine exert their action by acting directly on the peritoneal macrophage. Copyright (C) 2008 S. Karger AG, Basel.

Prolactin differentially modulates the macrophage activity of lactating rats: possible role of reproductive experience

Reproductive experience (i.e., pregnancy and lactation) induces physiological changes in mammals. We recently showed that a previous reproductive experience can modulate the activity of dopaminergic hypothalamic systems while decreasing serum prolactin (PRL) levels and oxidative burst activity in peritoneal macrophages. Dopamine receptor antagonists increase serum PRL levels, and both PRL and dopamine receptors might be involved in the modulation of macrophage activity, providing a means of communication between the nervous and immune systems. The present study evaluated the in vitro effects of PRL and the dopamine receptor 02 antagonist domperidone (DOMP) on the peritoneal activity of macrophages from primiparous and multiparous female rats during lactation. Oxidative bursts and phagocytosis in peritoneal macrophages were evaluated by flow cytometry. Primiparous and multiparous Wistar rats, during the period of lactation (i.e., days 5-7 after parturition) were used. Samples of peritoneal fluid from these rats were first incubated with PRL (10 and 100 nM) for different periods of time. The same procedure was repeated to evaluate the effects of DOMP (10 and 100 nM). Our results showed that macrophages from multiparous rats respond more effectively to in vitro incubation with PRL, especially with regard to oxidative bursts and the percentage of phagocytosis. Additionally, these effects were more pronounced after 30 min of incubation. These data suggest that reproductive experience is associated with a reduction in serum PRL levels, and cells in experienced female animals, including their macrophages, become more sensitive to the effects of PRL (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

The insulin hypoglycemia test: Hypoglycemic criteria and reproducibility

The insulin hypoglycemia test (IHT) is widely regarded as the 'gold standard' for dynamic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to investigate the temporal relationship between a rapid decrease in plasma glucose and the corresponding rise in plasma adenocorticotropic hormone (ACTH), and to assess the reproducibility of hormone responses to hypoglycemia in normal humans. Ten normal subjects underwent IHTs, using an insulin dose of 0.15 U/kg. Of these, eight had a second IHT (IHT2) and three went on to a third test (IHT3). Plasma ACTH and cortisol were measured at 15-min intervals and, additionally, in four IHT2s and the three IHT3s, ACTH was measured at 2.5- or 5-min intervals. Mean glucose nadirs and mean ACTH and cortisol responses were not significantly different between IHT1, IHT2 and IHT3. Combined data from all 21 tests showed the magnitude of the cortisol responses, but not the ACTH responses, correlated significantly with the depth and duration of hypoglycemia. All subjects achieved glucose concentrations of of less than or equal to 1.6 mmol/l before any detectable rise in ACTH occurred. In the seven tests performed with frequent sampling, an ACTH rise never preceeded the glucose nadir, but occurred at the nadir, or up to 15 min after. On repeat testing, peak ACTH levels varied markedly within individuals, whereas peak cortisol levels were more reproducible (mean coefficient of variation 7%). In conclusion, hypoglycemia of less than or equal to 1.6 mmol/l was sufficient to cause stimulation of the HPA axis in all 21 IHTs conducted in normal subjects. Nonetheless; our data cannot reveal whether higher glucose nadirs would stimulate increased HPA axis activity in all subjects. Overall, the cortisol response to hypoglycemia is more reproducible than the ACTH response but, in an individual subject, the difference in peak cortisol between two IHTs may exceed 100 nmol/l.

Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein- 1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

Exercise-induced alterations in neutrophil degranulation and respiratory burst activity: possible mechanisms of action

Neutrophils constitute 50-60% of all circulating leukocytes; they present the first line of microbicidal defense and are involved in inflammatory responses. To examine immunocompetence in athletes, numerous studies have investigated the effects of exercise on the number of circulating neutrophils and their response to stimulation by chemotactic stimuli and activating factors. Exercise causes a biphasic increase in the number of neutrophils in the blood, arising from increases in catecholamine and cortisol concentrations. Moderate intensity exercise may enhance neutrophil respiratory burst activity, possibly through increases in the concentrations of growth hormone and the inflammatory cytokine IL-6. In contrast, intense or long duration exercise may suppress neutrophil degranulation and the production of reactive oxidants via elevated circulating concentrations of epinephrine (adrenaline) and cortisol. There is evidence of neutrophil degranulation and activation of the respiratory burst following exercise-induced muscle damage. In principle, improved responsiveness of neutrophils to stimulation following exercise of moderate intensity could mean that individuals participating in moderate exercise may have improved resistance to infection. Conversely, competitive athletes undertaking regular intense exercise may be at greater risk of contracting illness. However there are limited data to support this concept. To elucidate the cellular mechanisms involved in the neutrophil responses to exercise, researchers have examined changes in the expression of cell membrane receptors, the production and release of reactive oxidants and more recently, calcium signaling. The investigation of possible modifications of other signal transduction events following exercise has not been possible because of current methodological limitations. At present, variation in exercise-induced alterations in neutrophil function appears to be due to differences in exercise protocols, training status, sampling points and laboratory assay techniques.

Present and future pharacotherapy for heart failure

The pharmacotherapy currently recommended by the American College of Cardiology and the American Heart Association for heart failure (HF) is a diuretic, an angiotensin-converting enzyme inhibitor (ACEI), a β-adrenoceptor antagonist and (usually) digitalis. This current treatment of HF may be improved by optimising the dose of ACEI used, as increasing the dose of lisinopril increases its benefits in HF. Selective angiotensin receptor-1 (AT1) antagonists are effective alternatives for those who cannot tolerate ACEIs. AT1 antagonists may also be used in combination with ACEIs, as some studies have shown cumulative benefits for the combination. In addition to being used in Stage IV HF patients, in whom it has a marked benefit, spironolactone should be studied in less severe HF and in the presence of β-blockers. The use of carvedilol, extended-release metoprolol and bisoprolol should be extended to severe HF patients as these agents have been shown to decrease mortality in this group. The ancillary properties of carvedilol, particularly antagonism at prejunctional β-adrenoceptors, may give it additional benefits to selective β1-adrenoceptor antagonists. Celiprolol and bucindolol are not the β-blockers of choice in HF, as they do not decrease mortality. Although digitalis does not reduce mortality, it remains the only option for a long-term positive inotropic effect, as the long-term use of the phosphodiesterase inhibitors is associated with increased mortality. The calcium sensitising drug levosimendan may be useful in the hospital treatment of decompensated HF to increase cardiac output and improve dyspnoea and fatigue. The antiarrhythmic drug amiodarone should probably be used in patients at high risk of arrhythmic or sudden death, although this treatment may soon be superseded by the more expensive implanted cardioverter defibrillators, which are probably more effective and have fewer side effects. The natriuretic peptide nesiritide has recently been introduced for the hospital treatment of decompensated HF. Novel drugs that may be beneficial in the treatment of HF include the vasopeptidase inhibitors and the selective endothelin-A receptor antagonists but these require much more investigation. However, disappointing results have been obtained in a large clinical trial of the tumour necrosis factor α antagonist etanercept, where no likelihood of a difference between placebo and etanercept was observed. Small clinical trials with recombinant growth hormone to thicken ventricles in dilated cardiomyopathy have given variable results.

Aerosolization of protein solutions using thermal inkjet technology

Vapotronics Inc. is developing the thermal inkjet (TIJ) technology used extensively in the printer industry to create a digital aerosol inhaler for the inhalation of therapeutics for local and systemic delivery. The operation of thermal inkjet printers requires generation of high temperatures and vaporization of the liquid formulation to effect droplet ejection. A study was conducted to develop formulations that would permit the generation of aerosols of therapeutic proteins without damage to the inkjet system or degradation of the proteins. Two proteins, human growth hormone and insulin, were formulated and aerosolized. The aerosol was collected and subjected to assays to compare the physicochemical and biological activities of these proteins before and after aerosolization. In each case, there was no significant changes to the proteins as a result of the aerosolization, providing evidence that TIJ can be used for aerosolizing solutions of protein therapeutics.

Classical approach to interpretation of the charge-dependence of peptide mobilities obtained by capillary zone electrophoresis

Published mobility measurements obtained by capillary zone electrophoresis of human growth hormone peptides are described reasonably well by the classical theoretical relationships for electrophoretic migration. This conformity between theory and experiment has rendered possible a more critical assessment of a commonly employed empirical relationship between mobility (u), net charge (z) and molecular mass (M) of peptides in capillary electrophoresis. The assumed linear dependence between u and z/M-2/3 is shown to be an approximate description of a shallow curvilinear dependence convex to the abscissa. An improved procedure for the calculation of peptide charge (valence) is also described. (C) 2003 Elsevier B.V. All rights reserved.

Hyperinsulinism and overgrowth without obesity

We report a 5 year old girl with postnatal overgrowth (height velocity >97th centile), hyperinsulinaemia, and increased insulin-like growth factor 1 for age, without evidence of bioactive or immunoreactive growth hormone excess or pituitary abnormality. Although her overgrowth may be a result of hyperinsulinism, her serum contains a factor (neither insulin nor IGF-1) which is able to stimulate the proliferation of lymphocyte precursors, and this could also account for the overgrowth. Over the course of two years observation she has developed acanthosis nigricans and diabetes mellitus.

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.

Long-term follow-up of four patients with Langer-Giedion syndrome: clinical course and complications.

Long-term observations of individuals with the so-called Langer-Giedion (LGS) or tricho-rhino-phalangeal type II (TRPS2) are scarce. We report here a on follow-up of four LGS individuals, including one first described by Andres Giedion in 1969, and review the sparse publications on adults with this syndrome which comprises ectodermal dysplasia, multiple cone-shaped epiphyses prior to puberty, multiple cartilaginous exostoses, and mostly mild intellectual impairment. LGS is caused by deletion of the chromosomal segment 8q24.11-q24.13 containing among others the genes EXT1 and TRPS1. Most patients with TRPS2 are only borderline or mildly cognitively delayed, and few are of normal intelligence. Their practical skills are better than their intellectual capability, and, for this reason and because of their low self-esteem, they are often underestimated. Some patients develop seizures at variable age. Osteomas on processes of cervical vertebrae may cause pressure on cervical nerves or dissection of cerebral arteries. Joint stiffness is observed during childhood and changes later to joint laxity causing instability and proneness to trauma. Perthes disease is not rare. Almost all males become bald at or soon after puberty, and some develop (pseudo) gynecomastia. Growth hormone deficiency was found in a few patients, TSH deficiency so far only in one. Puberty and fertility are diminished, and no instance of transmission of the deletion from a non-mosaic parent to a child has been observed so far. Several affected females had vaginal atresia with consequent hydrometrocolpos.

Adjuvant or radical fractionated stereotactic radiotherapy for patients with pituitary functional and nonfunctional macroadenoma.

Purpose: To evaluate the efficacy and toxicity of stereotactic fractionated radiotherapy (SFRT) for patients with pituitary macroadenoma (PMA).Methods and Materials: Between March 2000 and March 2009, 27 patients (male to female ratio, 1.25) with PMA underwent SFRT (median dose, 50.4 Gy). Mean age of the patients was 56.5 years (range, 20.3 - 77.4). In all but one patient, SFRT was administered for salvage treatment after surgical resection (transphenoidal resection in 23, transphenoidal resection followed by craniotomy in 2 and multiple transphenoidal resections in another patient). In 10 (37%) patients, the PMAs were functional (3 ACTH-secreting, 3 prolactinomas, 2 growth hormone-secreting and 2 multiple hormone-secretion). Three (11.1%) and 9 (33.3%) patients had PMA abutting and compressing the optic chiasm, respectively. Mean tumor volume was 2.9 +/- 4.6 cm(3). Eighteen (66.7%) patients had hypopituitarism prior to SFRT. The mean follow-up period after SFRT was 72.4 +/- 37.2 months.Results: Tumor size decreased for 6 (22.2%) patients and remained unchanged for 19 (70.4%) other patients. Two (7.4%) patients had tumor growth inside the prescribed treatment volume. The estimated 5-year tumor growth control was 95.5% after SFRT. Biochemical remission occurred in 3 (30%) patients with functional PMA. Two patients with normal anterior pituitary function before SFRT developed new deficits 25 and 65 months after treatment. The 5-year survival without new anterior pituitary deficit was thus 95.8%. Five patients with visual field defect had improved visual function and 1 patient with no visual defect prior to SFRT, but an optic chiasm abutting tumor, had a decline in visual function. The estimated 5-year vision and pituitary function preservation rates were 93.2% and 95.8%, respectively.Conclusions: SFRT is a safe and effective treatment for patients with PMA, although longer follow-up is needed to evaluate long-term outcomes. In this study, approximately 1 patient with visual field defect out of two had an improved visual.

Central and peripheral metabolic changes induced by gamma-hydroxybutyrate.

STUDY OBJECTIVES: Gamma-hydroxybutyrate (GHB) was originally introduced as an anesthetic but was first abused by bodybuilders and then became a recreational or club drug.1 Sodium salt of GHB is currently used for the treatment of cataplexy in patients with narcolepsy. The mode of action and metabolism of GHB is not well understood. GHB stimulates growth hormone release in humans and induces weight loss in treated patients, suggesting an unexplored metabolic effect. In different experiments the effect of GHB administration on central (cerebral cortex) and peripheral (liver) biochemical processes involved in the metabolism of the drug, as well as the effects of the drug on metabolism, were evaluated in mice. DESIGN: C57BL/6J, gamma-aminobutyric acid B (GABAB) knockout and obese (ob/ob) mice were acutely or chronically treated with GHB at 300 mg/kg. MEASUREMENTS AND RESULTS: Respiratory ratio decreased under GHB treatment, independent of food intake, suggesting a shift in energy substrate from carbohydrates to lipids. GHB-treated C57BL/6J and GABAB null mice but not ob/ob mice gained less weight than matched controls. GHB dramatically increased the corticosterone level but did not affect growth hormone or prolactin. Metabolome profiling showed that an acute high dose of GHB did not increase the brain GABA level. In the brain and the liver, GHB was metabolized into succinic semialdehyde by hydroxyacid-oxoacid transhydrogenase. Chronic administration decreased glutamate, s-adenosylhomocysteine, and oxidized gluthathione, and increased omega-3 fatty acids. CONCLUSIONS: Our findings indicate large central and peripheral metabolic changes induced by GHB with important relevance to its therapeutic use.