Significant Differences In Basal Levels Research Articles

Abstract B42: Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States. One of the major reasons for the poor prognosis of pancreatic cancer is its high resistance to gemcitabine and other currently available chemotherapeutic agents. In the present study, we have developed gemcitabine-resistant cell lines to investigate the mechanisms by which adaptive resistance occurs during pancreatic tumor progression. Gemcitabine-resistant (GR) cell lines were established from the MiaPaCa2 cell line. Increasing drug selection pressure resulted in the establishment of populations that proliferate in the continuous presence of 300 nm (GR300), 800 nM (GR800), or 2µM (GR2000) gemcitabine. Using a SCID mouse xenograft mouse model, we demonstrate that the gemcitabine resistant phenotype is maintained in vivo. Growth inhibition profiles demonstrate cross resistance to Ara-c (cytarabine), and 2-chlorodeoxyadenosine (2CDA/cladribine), but no resistance to DNA damaging agents or microtubule inhibitors. Most strikingly, the gemcitabine resistant subclones demonstrated a dose dependent hypersensitivity to the Class I histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) (IC50: MP 1.6uM; GR300 1.0uM; GR800 0.2uM; GR2000 0.08uM). This collateral hypersensitivity extended to additional Class I HDAC inhibitors including MS-275 (Entinostat) and Trichostatin A, but not to the Class IIa selective agent MC1568. There was no significant difference in basal levels of histone acetylation, but the gemcitabine resistant clones demonstrated a greater increase in histone acetylation in response to HDAC treatment. Cell morphology of the gemcitabine resistant cells demonstrate an EMT-like phenotype, however evaluation of classic EMT markersidentified no significant changes in expression compared to the parental cell line. Invasive potential was measured using matrigel invasion assays, and found to be increased in a dose dependent manner. These data demonstrate that selection for drug resistance co-selects for an invasive phenotype with EMT-like characteristics. In addition, the resistance phenotype that emerges under drug pressure is associated with a phenotypic reprogramming that can be exploited to devise new therapeutic options for patients with advanced pancreatic adenocarcinoma. Citation Format: Betty k. Samulitis, Erika Pond, Kelvin Pond, Anne E. Cress, Robert T. Dorr, Terry H. Landowski. Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B42.

Caudate nucleus-dependent response strategies in a virtual navigation task are associated with lower basal cortisol and impaired episodic memory

The present research examined the relationship between endogenous glucocorticoids, navigational strategies in a virtual navigation task, and performance on standard neuropsychological assessments of memory. Healthy young adult participants (N=66, mean age: 21.7) were tested on the 4 on 8 virtual maze (4/8 VM) and standard neuropsychological tests such as the Rey-Osterrieth Complex Figure (RO) and the Rey Auditory Verbal Learning Task (RAVLT), which measure episodic memory. The 4/8 VM differentiates between navigational strategies, where participants either use a hippocampal-dependent spatial strategy by building relationships between landmarks, or a caudate nucleus-dependent stimulus–response strategy by automatizing a pattern of open and closed arms to learn the location of objects within the maze. Degree of stress was assessed by administering the Perceived Stress Scale (PSS) questionnaire. Cortisol samples were taken on two consecutive days upon waking, 30min after waking, at 11am, 4pm, and 9pm. There was a significant difference in basal levels of cortisol between spatial and response learners. Interestingly, response learners had significantly lower cortisol levels throughout the day. The two groups did not differ in terms of perceived stress as measured with the PSS questionnaire. Moreover, there was no significant correlation between PSS scores and salivary cortisol levels, indicating that the higher cortisol levels in the spatial group were not associated with greater perceived stress. In addition, participants who spontaneously used a spatial strategy performed significantly better on the RAVLT and RO. These data indicate that the cortisol levels in the spatial group may be optimal in terms of episodic memory performance whereas the cortisol levels in the response group may be associated with poorer memory. These results are suggestive of an inverted U-shaped curve describing the effects of basal levels of circulating cortisol on memory in young adults.

A new method for producing urinary bladder hyperactivity using a non-invasive transient intravesical infusion of acetic acid in conscious rats

Introduction Animal models that closely resemble the pathophysiology of human overactive bladder are important for evaluating novel therapeutics to treat the disorder. We established a non-invasive hyperactive bladder model that is sensitive to anti-muscarinic drugs and without bladder inflammation. Methods Acetic acid solution was infused into the bladder for 5 min via the urethral orifice without any surgical procedures under isoflurane anaesthesia. After washing the bladder with saline, voiding frequency (VF) and total urine volume were determined for 9 h under conscious conditions. Results Infusion of a 0.5% acetic acid solution caused a significant increase in VF, without influencing total urine volume or inducing significant histopathological inflammatory alterations in the bladder urothelium. Oral administration of oxybutynin (3 and 10 mg/kg) significantly ameliorated increases in VF induced by 0.5% acetic acid. Infusion of 0.75% acetic acid induced intensive urinary inflammation and a decrease in total urine volume as well as an increase in VF. Oral treatment with oxybutynin (10 mg/kg) did not significantly improve the increased VF due to 0.75% acetic acid. Acetic acid (0.5%) infusion evoked bladder hyper-responsiveness whether applied at night or during the day. However, VF was increased more by the nighttime application of acetic acid, while there were no significant differences in basal levels of VF between daytime and nighttime. Discussion In this study, the non-invasive rat urinary hyperactive bladder model indicated minimizes the secondary effects of experimental procedures such as surgical operations and anesthesia on bladder function and is sensitive to oxybutynin. Thus, the model may be useful for investigating novel therapeutics for OAB treatment.

Requirement of Vimentin Filament Assembly for β3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.

Increased resistance of GPx-1 transgenic mice to tumor promoter-induced loss of glutathione peroxidase activity in skin.

Reactive oxygen species formation is strongly suspected to play a role in multistep carcinogenesis, notably in tumor promotion. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces peroxide production, oxidative damage to DNA and inflammation in mouse skin. TPA is also known to cause a decrease in the activity of several antioxidant enzymes including glutathione peroxidases (GPx). The observation that several anti-oxidants can inhibit TPA-mediated tumor promotion suggests that a decline in GPx activity could contribute to tumor promotion. We report here the effects of TPA on GPx activity in the skin of transgenic GPx mice that contain human GPx-1 transgenes under the regulation of a metallothionein IIA promoter. As expected, no significant difference in basal level of skin GPx activity was detected in the 3 lines of tg-MT-GPx mice investigated compared with non-transgenic controls. A single topical application of TPA induced gradually, over 20 hr, a small but detectable increase in GPx mRNA and protein levels in skin of non-transgenic mice and a contrasting decrease in both selenium-dependent and selenium-independent GPx activity. The extent of GPx induction was more pronounced in transgenic mice, and in contrast with non-transgenic mice, no significant loss of GPx activity was observed in the TPA-treated skin of these mice. Transgenic mice may, therefore, offer a novel model suitable to assess the role of GPx-1 in skin carcinogenesis, without the potential disadvantage of abnormally high levels of GPx activity produced constitutively in other transgenic models.

Differential effects of chronic imipramine and fluoxetine on basal and amphetamine-induced extracellular dopamine levels in rat nucleus accumbens

The effect of chronic treatment with the tricyclic antidepressant drug, imipramine (10 mg/kg per day), the selective serotonin (5-HT) reuptake inhibitor, fluoxetine hydrochloride (10 mg/kg per day), and vehicle, in drinking water for 24–28 days followed by 3–5 days withdrawal, on extracellular dopamine levels was studied in rat nucleus accumbens by in vivo microdialysis. Basal extracellular dopamine levels in the nucleus accumbens were increased after chronic imipramine (12.7±1.5 fmol/20 μl per 30 min, P=0.019), and moderately decreased after chronic fluoxetine (6.5±0.6, P=0.047), as compared to the vehicle controls (9.1±0.7), determined by one-way analysis of variance (ANOVA). Repeated measure ANOVA indicated that the d-amphetamine sulfate (0.5 mg/kg, s.c.)-induced increase in extracellular dopamine levels in the nucleus accumbens was potentiated after chronic imipramine ( P=0.002), but unchanged after chronic fluoxetine ( P=0.83). The difference in the effect of amphetamine could be influenced by the significant differences in basal levels. However, these results were also confirmed by analysis of the net area under the curve (net-AUC) for a 180-min period (six samples): for chronic imipramine (337±45 fmol/180 min, P=0.005) and chronic fluoxetine (249±38, P=0.57), as compared to the vehicle controls (178±29), determined by one-way ANOVA. We suggest that the effect of treatment with these agents on mesolimbic dopamine is unlikely to be involved in their shared antidepressant action, but may be relevant to other aspects of the therapeutic profile of these two drugs, e.g. the switch into mania which is more common after treatment with imipramine than fluoxetine and exacerbation of positive symptoms in patients with schizophrenia or schizoaffective disorder.

Bone density changes in postmenopausal women with the administration of ipriflavone alone or in association with low-dose ERT

Ipriflavone is a synthetic flavonoid that has been shown to exert a direct inhibitory effect on osteoclastic activity and possibly stimulate the osteoblast activity in different experimental models. The aim of the present study was to evaluate the effects of either ipriflavone alone or ipriflavone plus low dose hormone replacement therapy (HRT) in the prevention of postmenopausal bone loss. Patients were randomly allocated to different treatment groups receiving calcium (500 mg/day, control group), continuous HRT (25 or 50 μg/day of transdermal 17β-estradiol) plus medrogestone 5 mg/day for 12 days/month, ipriflavone at the standard dose of 600 mg/day, or finally ipriflavone 600 mg/day plus 17β-estradiol 25 μ g/day plus medrogestone 5 mg/day for 12 days/month. No significant differences in basal levels of biochemical markers of bone turnover or in basal bone mineral density (BMD) values were evident in the different groups. In the control group after 12 months, spine BMD showed a significant (p < 0.05) 3.41% decrease. The pattern of BMD modification was significantly different from controls in the high dose HRT group (+ 1.84%), the ipriflavone group (+ 0.11%), and the combined ipriflavone/HRT group (-0.22%). Conversely, the BMD pattern in the low dose HRTgroup (-0.55%) was similar to that observed in controls. Thus, present results confirm that ipriflavone and 50 μg/day of transdermal 17β-estradiol are effective measures in the prevention of postmenopausal osteopenia. A lower transdermal estrogen dose is unable to increase the antiresorptive effect of ipriflavone and did not exert any further action in the prevention of postmenopausal osteopenia.

Micronized estradiol and progesterone: Effects on carbohydrate metabolism in reproductive-age women

We assessed glucose utilization and insulin sensitivity in normal reproductive-age women after administration of micronized estradiol, micronized progesterone, or the combination of micronized estradiol and progesterone. Hyperglycemic and euglycemic, hyperinsulinemic clamp studies were performed in normal, regularly cycling women both before and after a short course of either micronized estradiol (n = 8), micronized progesterone (n = 8), or micronized estradiol and progesterone (n = 7). All studies were performed after an overnight fast. Glucose and insulin were determined in the control period and then every 2-10 minutes in the hyperglycemic clamp studies and every 5-10 minutes in the euglycemic clamp studies. In the hyperglycemic clamp studies, hyperglycemia was maintained by a variable glucose infusion. In the euglycemic clamp studies, a primed 3-3H glucose infusion (25 microCi) followed by a 0.25-microCi continuous infusion was begun 120 minutes before initiation of the insulin infusion and variable glucose infusion. Samples for glucose radioactivity were measured during the control period and during the last 40 minutes of each step of the euglycemic clamp after establishment of a steady state. No differences in baseline glucose or insulin levels were detected in any of the study groups as compared with control. Glucose utilization as assessed by the hyperglycemic clamp model was not significantly different from control in the estradiol group, the progesterone group, or the group treated with the combination. In all of the treatment groups, no significant differences were noted under euglycemic, hyperinsulinemic conditions in glucose utilization, hepatic glucose production, or insulin sensitivity. Women of reproductive age do not demonstrate significant differences in basal levels of glucose or insulin when given a short course of micronized estradiol and progesterone, either alone or in combination. Under conditions of the hyperglycemic, hyperinsulinemic clamp, the pancreatic beta-cell response to hyperglycemia and glucose utilization is not significantly altered by exogenous administration of hormones. Conditions of the euglycemic, hyperinsulinemic clamp failed to elicit significant differences in glucose utilization and insulin sensitivity in any of the three treatment groups. These findings demonstrate that glucose homeostasis is not altered by the exogenous administration of natural hormones in reproductive-age women.

An effect of somatostatin on neurotensin secretion

A somatostatin analog (SMS201-995) was administered subcutaneously to six totally pancreatectomized (TP) patients at five dose levels from 0 μg/kg (control) to 2.5 μg/kg before ingestion of a liquid test meal, and plasma levels of neurotensin (NT) were measured. There were no significant differences in basal levels of NT between the doses of somatostatin. The NT levels were significantly elevated after ingestion of the test meal in the normal controls ( n = 14). In the TP patients postprandial hypersecretion of NT was observed. The hypersecretion of NT in the patients was suppressed by SMS201-995 dose-dependently.

Pancreatic insulin secretory response and insulin action in heat-exposed sheep given a concentrate or roughage diet

The effects of heat exposure and type of diet on the insulin secretory response to glucose and glucose disposal in response to insulin action in female sheep were investigated employing hyperglycemic and euglycemic clamp techniques. Animals were divided into concentrate and roughage diet groups, and were maintained at the same intake levels of metabolizable energy and crude protein in both diets. Each diet group was subjected to either thermoneutral (20°C, 70% RH) or hot (30°C, 70% RH) environment, followed by glucose clamp experiments. Heat-exposed sheep showed significant increases in respiration rates (P<.001) and rectal temperature (P<.05). Plasma glucose concentrations in the basal conditions were lower (P<.01) in the hot environment than in the thermoneutral environment, but there was no significant difference in basal levels of plasma insulin between the environmental treatments. In the hyperglycemic clamp experiment, mean plasma insulin increments increased (P<.05) during the heat exposure period across diet treatments. The ratio of mean plasma insulin increment to glucose infusion rate tended to be higher (P<.07) in the hot environment than in the thermoneutral environment, but diet treatment did not affect the ratio of mean plasma insulin increment to glucose infusion rate. The euglycemic clamp technique showed that glucose infusion rates remained unchanged among treatments. Insulin secretion response to glucose could be stimulated in the hot environment.

Phosphate Depletion Reduces Potassium-Induced Insulin Secretion

Potassium-induced insulin secretion is impaired in rats with chronic renal failure and a sustained rise in cytosolic calcium ([Ca2+]i). It has been found that the calcium signal (delta[Ca2+]i) and the delta [Ca2+]i/basal [Ca2+]i in these animals in response to potassium are smaller than those in normal rats and that these defects may underlie, at least in part, the reduced potassium-induced insulin secretion, since the latter depends on an appropriate rise in [Ca2+]i. Since phosphate depletion (PD) is another model associated with a rise in the basal level of [Ca2+]i of pancreatic islets, it provides another metabolic setting for investigating the interaction between high [Ca2+]i of islets and their response to potassium. We examined the potassium-induced insulin secretion, the potassium-induced calcium signal, and the delta [Ca2+]i/basal [Ca2+]i in islets of PD rats with and without elevated [Ca2+]i. The levels of the basal [Ca2+]i in the islets of PD rats were significantly (P less than 0.01) higher than those in pair-weighed (PW) animals and those in PD and PW rats treated with verapamil, which has been shown to prevent the rise in [Ca2+]i in islets of PD rats. Both initial and total insulin secretion, the calcium signal, and the delta [Ca2+]i/basal [Ca2+]i in the islets of PD rats were significantly (P less than 0.01) smaller than those in the other three groups of animals. There were no significant differences in basal levels of [Ca2+]i and in calcium signal, delta [Ca2+]i/basal [Ca2+]i, and insulin secretion among PW rats, verapamil-treated PD rats, and verapamil-treated PW rats. The results are consistent with the notion that elevated resting levels of [Ca2+]i interfere with the magnitude of the calcium signal and the ratio of calcium signal to basal [Ca2+]i, and these derangements, at least in part, underlie the impaired potassium-induced insulin secretion in PD.

Acute effects of calcitriol and phosphate salts on mineral metabolism in children with hypophosphatemic rickets

We investigated the acute effects of oral administration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) and phosphate on the major mineral metabolism indexes in six children with vitamin D-resistant rickets treated with a long-term regimen of phosphate and calcitriol. Two acute tests were performed in which plasma calcium, phosphate, immunoreactive parathyroid hormone (iPTH) (intact molecule), 25-hydroxyvitamin D (25-OHD), and 1,25-(OH)2D levels were measured: the first after an oral phosphate load (20 mg/kg) was administered after calcitriol had been discontinued for 10 days, and the second after a calcitriol load (0.03 microgram/kg) plus the same phosphate load but with the children receiving the usual combination treatment. There were no significant differences in basal levels of calcium, phosphate, iPTH, 25-OHD, or 1,25-(OH)2D between the two tests, nor were delta percent calcium and 25-OHD values significantly different. The delta percent plasma phosphate concentration at 60 minutes was significantly higher during test 2 than during test 1 (p less than 0.01) and delta percent iPTH concentration at 60 minutes was significantly higher during test 1 than during test 2 (p less than 0.01). In test 2 the iPTH level returned to baseline at 180 minutes. Higher delta percent 1,25-(OH)2D values at 60 minutes were observed in test 2 than in test 1 (p less than 0.01). Furthermore, the delta percent 1,25-(OH)2D levels were still higher at 180 minutes in test 2 than during test 1 (p less than 0.01). Our study indicates that oral calcitriol has an inhibitory effect on iPTH secretion in the hours immediately after oral phosphate administration in children with vitamin D-resistant rickets.

MINIMAL THYROID INSUFFICIENCY(MTI) IN CHILDREN WITH CONSTITUTIONAL DELAYED GROWTH(CDG)

Therapeutic trial c thyroid preparation in absence of clinical & laboratory evidence of hypothyroidism has been reported in children c short stature. (J.Ped. 80,988,72). Patients c MTI have normal T3,T4,& TSH levels but an augmented TSH response to TRH. (Peak TSH response > 20uIU/ml). TSH & T3 response to TRH was assessed in 20 children (mean age 11.7 range 7.9-15.8 yrs ♂ 18: ♀ 2) c CDG. TRH test was done with IV bolus TRH 4ug/kg/BW. after an overnight fast of 10 hrs. 5 had augmented TSH response (Gr.B) 15 had normal (Gr.A). There was no significant difference in basal levels of T3,T4,free T4 & TSH. Mean TSH reponse was significantly higher at all times in Gr.B vs Gr.A T3 % increase was lower in Gr.B 29.1±2.5(N3) vs Gr.A 41.9±4.0(N9) p < 0.05. Augmented TSH response & lower T3 % increase in response to TRH was found in 25% of children c CDG. This could be an evidence of minimal thyroid insufficiency or of hypothalamic pituitary thyroid dysfunction in children c CDG. Treatment c thyroxine in two patients for 5 months showed 140% increase in annual growth rate. Controlled therapeutic trial is indicated.

THYROTROPIN (TSH) RESPONSE TO THYROTRUPIN RELEASING HORMONE (TRH) IN CHILDREN &amp; ADOLESCENTS WITH OBESITY

A number of abnormalities in hypothalamic pituitary functions have been reported in non-endocrine obesity. No reports are available on hypothalamic pituitary-thyroid status in children with obesity.TSH response to TRH was studied in 47 obese patients (Gr.A) (mean age 12.4±0.5 range 5.1-19.2 yrs; ♂ 25, ♀ 22) & was compared with 24 short-normal controls (Gr.B) (mean age 12.3±0.6 range 5.2-17.5 yrs; ♂ 18, ♀ 6) Mean % overweight 64.8±6.1 range 11.3-226.0.TRH test was done with IV bolus 4ug/kg/BW (maximum 250 ug) of TRH after overnight fast of 10 hrs. There was no significant difference in basal levels of T4,T3, free T4 & TSH. 34.0% of Gr.A had augmented TSH response (Peak TSH > 20uIU/ml). Mean TSH response was significantly higher in Gr.A vs Gr.B.The higher/augmented TSH response to TRH in obese children could be a evidence of hypothalamic pituitary dysfunction, of partial peripheral resistance to thyroid hormone, or of minimal thyroid insufficiency.

Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation.

The adaptations in the freshwater turtle that permit survival despite prolonged loss of mitochondrial ATP generation were investigated by comparing the bioenergetics of turtle brain slices with rat brain slices. Aerobic turtle brain shows no significant difference in basal levels of total ATP generation compared to rat brain; levels in turtle brain and rat brain were 18.4 +/- 2.8 (SD) and 19.4 +/- 2.2 mumol (100 mg of tissue)-1 hr-1, respectively. However, in turtle brain, a significantly greater fraction of ATP is derived from glycolysis both under aerobic and anaerobic conditions [aerobic turtle (24%) and rat (13%), P less than 0.02; anaerobic, turtle (28%) and rat (18%), P less than 0.05]. The increased glycolytic capacity is related to high levels of rate-limiting glycolytic enzymes, such as pyruvate kinase (EC 2.7.1.40). Turtle brain operates close to glycolytic capacity even under aerobic conditions, and no Pasteur effect can be demonstrated. Quantitatively, anaerobic glycolysis accounts for a maximum of 28% of basal aerobic ATP generation, suggesting that prolonged diving is also accompanied by a reduction in brain energy requirements. The adaptation subserving short-term (natural) diving is an increase in brain glycolytic capacity. The adaptation subserving prolonged diving (days to weeks) may be a reduction in the energy requirements of brain (and other cells).

Mechanisms of Puerperal Lactation

During pregnancy estrogen-medicated augmented prolactin secretion is presumably responsible for a 10-20 fold increase in circulating plasma prolactin; significant differences in basal levels between nursing and nonnursing women persist into the puerperium, reflecting the influence of sucking on maternal plasma prolactin. The release of prolactin is induced via a neurogenic pathway from nipple to hypothalamus and it is proportionate to the length of nursing and to the intensity of the stimulus. There is evidence supporting catecholamine/serotonin control of prolactin release, and the influence of changes in hypothalamic dopamine turnover. The composition of human milk is dependent on various factors; overall, fat composition is 2-5% and protein 9% at 3 weeks and 5% thereafter; milk delivers 20-25 calories per ounce; total fluid and nutritional requirements of the newborn can be met by breastfeeding up to 6 months postpartum. Maternal malnutrition negatively affects lactation; gestational, rather than progestational, food intake influences lactation. Immunity in the newborn is provided also by breast milk through immunoglobulins, thus enhancing the child's protection against internal pathogens. The incidence of gastrointestinal disorders is 1.5/1000 in breastfed infants, and 84.7/1000 in bottle fed infants; the incidence of respiratory infection is .4/1000 and 48/1000, respectively. Prolactin may exert an inhibitory influence on ovarian steroidogenesis, and gonadotropin secretion is disrupted by nipple stimuation; this may account for the low percentage of ovulation among nursing mothers. Lactational amenorrhea has been proven to have great demographic impact; dramatic variations in fertility on the basis of variations in lactational amenorrhea have been described in rural areas of Latin America, Asia, and Africa. Reduction of lactational amenorrhea results not only from changes in sociocultural patterns, but from improved maternal nutrition, often through nutrition programs. When nursing has to be interrupted because of complications full lactation may be restored by oral administration of thyrotropin-releasing hormone. Breastfeeding is possible in 99% of women; the denial of lactation may cause the retention of unwanted weight, which can be compounded by the use of oral contraceptives. Moreover, infantile obesity may stem from the lack of a satiety signal in bottle fed newborns.