Childbirth and consequent atopic disease: emerging evidence on epigenetic effects based on the hygiene and EPIIC hypotheses.

BACKGROUND: In most high and middle income countries across the world, at least 1:4 women give birth by cesarean section. Rates of labour induction and augmentation are rising steeply; and in some countries up to 50% of laboring women and newborns are given antibiotics. Governments and international agencies are increasingly concerned about the clinical, economic and psychosocial effects of these interventions. DISCUSSION: There is emerging evidence that certain intrapartum and early neonatal interventions might affect the neonatal immune response in the longer term, and perhaps trans-generationally. Two theories lead the debate in this area. Those aligned with the hygiene (or 'Old Friends') hypothesis have examined the effect of gut microbiome colonization secondary to mode of birth and intrapartum/neonatal pharmacological interventions on immune response and epigenetic phenomena. Those working with the EPIIC (Epigenetic Impact of Childbirth) hypothesis are concerned with the effects of eustress and dys-stress on the epigenome, secondary to mode of birth and labour interventions. This paper examines the current and emerging findings relating to childbirth and atopic/autoimmune disease from the perspective of both theories, and proposes an alliance of research effort. This is likely to accelerate the discovery of important findings arising from both approaches, and to maximize the timely understanding of the longer-term consequences of childbirth practices.

The EPIIC hypothesis: intrapartum effects on the neonatal epigenome and consequent health outcomes.

There are many published studies about the epigenetic effects of the prenatal and infant periods on health outcomes. However, there is very little knowledge regarding the effects of the intrapartum period (labor and birth) on health and epigenetic remodeling. Although the intrapartum period is relatively short compared to the complete perinatal period, there is emerging evidence that this time frame may be a critical formative phase for the human genome. Given the debates from the National Institutes of Health and World Health Organization regarding routine childbirth procedures, it is essential to establish the state of the science concerning normal intrapartum epigenetic physiology. EPIIC (Epigenetic Impact of Childbirth) is an international, interdisciplinary research collaboration with expertise in the fields of genetics, physiology, developmental biology, epidemiology, medicine, midwifery, and nursing. We hypothesize that events during the intrapartum period - specifically the use of synthetic oxytocin, antibiotics, and cesarean section - affect the epigenetic remodeling processes and subsequent health of the mother and offspring. The rationale for this hypothesis is based on recent evidence and current best practice.

The P2X7-Egr pathway regulates nucleotide-dependent inflammatory gene expression in microglia.

Microglial hyperactivity contributes to neuronal damage resulting from CNS injury and disease. Therefore, a better understanding of endogenous microglial receptor systems that can be exploited to modulate their inflammatory functions is important if better, neuroprotective therapeutics are to be designed. Previous studies from our lab and others have demonstrated that the P2X7 purinergic receptor agonist BzATP attenuates microglial inflammatory mediator production stimulated by lipopolysaccharide (LPS), suggesting that purinergic receptors may be one such receptor system that can be used for manipulating microglial activation. However, although P2X7 receptor activation is well recognized to regulate processing and release of cytokines, little is known concerning its role in regulating the transcription of inflammatory genes, nor the molecular mechanisms underlying these transcriptional effects. In the present studies, we identify that the transcription factors early growth response (Egr)-1, -2 and -3 are downstream signaling targets of P2X7 receptors in microglia, and that their activation is sensitive to MEK and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, using RNAi, we demonstrate that Egr factors and P2X7 receptors are necessary for BzATP-mediated attenuation of iNOS, and stimulation of TNF-α and IL-6 gene expression. BzATP also attenuates neuronal death induced by LPS conditioned medium, and P2X7 receptors are required for this effect. These studies are the first to identify Egr factors as regulators of inflammatory gene expression following P2X7 receptor activation, and suggest that P2X7 receptors may utilize the MAPK-Egr pathway to exert differential effects on microglial inflammatory activities which are beneficial to neuron survival.

Location and type of mutation in the LIS1 gene do not predict phenotypic severity.

BACKGROUND: Lissencephaly is a neuronal migration disorder leading to absent or reduced gyration and a broadened but poorly organized cortex. The most common form of lissencephaly is isolated, referred as classic or type 1 lissencephaly. Type 1 lissencephaly is mostly associated with a heterozygous deletion of the entire LIS1 gene, whereas intragenic heterozygous LIS1 mutations or hemizygous DCX mutations in males are less common. METHODS: Eighteen unrelated patients with type 1 lissencephaly were clinically and genetically assessed. In addition, patients with subcortical band heterotopia (n = 1) or lissencephaly with cerebellar hypoplasia (n = 2) were included. RESULTS: Fourteen new and seven previously described LIS1 mutations were identified. We observed nine truncating mutations (nonsense, n = 2; frameshift, n = 7), six splice site mutations, five missense mutations, and one in-frame deletion. Somatic mosaicism was assumed in three patients with partial subcortical band heterotopia in the occipital-parietal lobes or mild pachygyria. We report three mutations in exon 11, including a frameshift which extends the LIS1 protein, leading to type 1 lissencephaly and illustrating the functional importance of the WD domains at the C terminus. Furthermore, we present two patients with novel LIS1 mutations in exon 10 associated with lissencephaly with cerebellar hypoplasia type a. CONCLUSION: In contrast to previous reports, our data suggest that neither type nor position of intragenic mutations in the LIS1 gene allows an unambiguous prediction of the phenotypic severity. Furthermore, patients presenting with mild cerebral malformations such as subcortical band heterotopia or cerebellar hypoplasia should be considered for genetic analysis of the LIS1 gene.

The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana (bullfrog) tadpoles is induced by thyroxine.

Melatonin decreases in the plasma of Rana catesbeiana (bullfrog) tadpoles at the climax of metamorphosis when the thyroxine (T(4)) level peaks. Since melatonin inhibited thyroid function in vitro, it would be of interest to determine if the decline in plasma melatonin permits greater thyroid hormone secretion, or if the increasing levels of T(4) cause the climactic decrease in plasma melatonin. The reciprocal effects of administering T(4) or melatonin just prior to metamorphic climax were examined in tadpoles kept at 22 degrees C on an 18L:6D cycle. If melatonin functions as a thyroid antagonist at later metamorphic stages, administration of melatonin should decrease plasma T(4), whereas if T(4) causes the decline in plasma melatonin, T(4) treatment of tadpoles prior to climax should induce the climactic melatonin decrease prematurely. Once daily injection of 40 microg melatonin for 5 days at 19.30 h had no effect on metamorphic progress, or on plasma T(4) or melatonin levels, except for a transient rise in melatonin just after the injection. Immersion in 2.2x10(-4) M melatonin for 6 days accelerated metamorphosis and decreased plasma melatonin, but had no effect on plasma T(4). Administration of T(4) by injection of 0.2 microg, or immersion in a 6.3x10(-8) M solution accelerated metamorphosis more than melatonin immersion, raised plasma T(4) to climax levels, and induced a decrease in plasma melatonin. We conclude that rapid clearance of exogenous melatonin from the circulation in these experiments did not allow it to affect plasma T(4), and that there is clear evidence that the rise in T(4) induces the climax decrease in plasma melatonin. The finding that immersion in a high level of melatonin can lower plasma melatonin and accelerate metamorphosis, whereas a single daily injection does not, provides an explanation for some of the contradictory reports in the literature concerning melatonin's effect on tadpole metamorphic progress.

Direct influence of melatonin on the thyroid and comparison with prolactin.

Melatonin administered in vivo had previously been shown to inhibit thyroid cell proliferation and subsequent in vitro thyroxine (T(4)) secretion in anuran tadpoles. Melatonin in vitro also directly reduced the sensitivity of the thyroid to thyrotropin (TSH). The present work sought to determine whether melatonin directly affected baseline, unstimulated T(4) secretion, and to compare its effect with that of prolactin (PRL). Thyroids from larval Rana catesbeiana or adult Rana pipiens were incubated in control or melatonin (0.01 to 100 microg/ml) media. Melatonin directly inhibited T(4) secretion by thyroids from both tadpoles and frogs at all concentrations of melatonin used and at both prometamorphic and climax tadpole stages. PRL, used in vitro at 10 microg/ml, did not influence the response of the thyroid to TSH (0.2 microg/ml) in young tadpoles, or the baseline secretion of T(4) by thyroids at any stage of larval life except climax, when T(4) secretion was significantly decreased by the third day of culture. Thus although both melatonin and PRL have been shown to antagonize the action of T(4) in vitro, and to decrease metamorphic rate, melatonin is a much more effective thyroid gland inhibitor than PRL.

Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles.

Bullfrog (Rana catesbeiana) tadpoles are of value to amphibian researchers because of their large size, and year-round availability due to overwintering in many latitudes. Concern over a possible difference in hormonal parameters in tadpoles obtained at different times of the year prompted us to investigate thyroid gland secretion in vitro, plasma and ocular melatonin, and plasma corticosteroids in late pre- to early prometamorphic larvae on 12L:12D. Winter tadpoles exposed to 22 degrees C for 3 weeks of acclimation (winter group) were compared to summer tadpoles kept at 22 degrees C (summer group), as well as to summer tadpoles exposed to cold (12 degrees C) for the 3 weeks (cold group), or kept at 22 degrees C and starved for the last week of acclimation (starved group). Thyroids from the summer group had a significantly higher response to 0.2 microg/ml ovine thyrotropin (TSH) than the other groups, indicating that cold and starvation inhibited subsequent in vitro thyroid sensitivity to TSH. The thyroids of the starved tadpoles had significantly higher baseline (unstimulated) thyroxine (T4) secretion into the culture media, a finding that might be related to starvation-induced acceleration of metamorphosis. Plasma melatonin was lower, and ocular melatonin significantly higher in both summer and starved groups, while the reverse occurred in the winter and cold groups. Thus, seasonal or induced cold brought on a shift in the relationship of plasma to ocular melatonin but starvation had no effect. There were no significant differences among the treatment groups in plasma hydrocortisone (HC) and aldosterone (ALDO) levels, except that HC was lower than ALDO only in the plasma of winter tadpoles. We conclude that seasonal variation needs to be taken into account in endocrine experiments utilizing tadpoles obtained at different times of the year.

Influence of cortisol on the larval bullfrog thyroid axis in vitro and in vivo and on plasma and ocular melatonin.

Adrenal (interrenal) steroids have an important role in amphibian development, antagonizing the metamorphic changes induced by the thyroid at first and then synergizing with the thyroid hormones as their level rises during metamorphosis. Because most of the studies of corticoids at metamorphosis have focused on peripheral tissues, we investigated the effect of cortisol (hydrocortisone; HC) in vitro and in vivo on the thyroid of Rana catesbeiana (bullfrog) tadpoles on 12:12 light/dark (LD) cycles. Plasma and ocular melatonin, which is altered by changes in thyroxine (T(4)) levels, were also assayed in some experiments. Thyroids from premetamorphic tadpoles secreted less T(4) into culture media when incubated with 10 micrograms/ml HC and 0.2 micrograms/ml ovine thyrotropin (TSH) than with TSH alone and when cultured in the absence of TSH following 5 days of 10-micrograms HC injections, indicating that HC inhibited the thyroid at young stages. The effect of 10 micrograms/ml HC at older stages was investigated by culturing thyroids and pituitaries separately on the first day in control or HC media and then incubating the thyroids on the second day in homologous pituitary-conditioned media as a bioassay for pituitary TSH. HC had no effect on baseline T(4) secretion by the thyroids of prometamorphic or climax tadpoles on the first day but increased T(4) secretion over the control on the second day. Thyroids cultured with TSH and HC showed no increase in T(4) secretion over the control TSH group on the second day, indicating that, in the previous experiments, HC had enhanced pituitary secretion of TSH, rather than the response of the thyroid to TSH. In vivo, 5 days of injections of 10 micrograms HC increased plasma T(4) at prometamorphosis and decreased it at climax. There was no marked effect of HC on plasma or ocular melatonin levels. The findings showed that the nature of the effect of HC on the thyroid axis changes during metamorphosis from inhibition at early stages to a positive influence at prometamorphosis and finally to a negative effect on the T(4) level in the plasma at climax.

Distribution and reciprocal interactions of 3H-melatonin and 125I-thyroxine in peripheral, neural, and endocrine tissues of bullfrog tadpoles.

Tissue distribution of 125I-thyroxine (T4) and 3H-melatonin and the effect of each hormone on the tissue content of the other were studied because previous work indicated that melatonin antagonized metamorphosis through peripheral, as well as thyroidal effects. Late pre- to prometamorphic Rana catesbeiana tadpoles on an 18 light:6 dark cycle were used for injection of hormones in vivo or to supply tissues for in vitro hormone administration. Labeled melatonin uptake was highest in intestine, ventral skin and pituitary; lowest in thyroid and brain and intermediate in hindlimb, tail and gills. The tissue content of labeled T4 was distributed in nearly the same way, except that the thyroid level was relatively higher, and pituitary lower, than that of labeled melatonin. The pineal, studied only in the tracer T4 experiments, had the highest content of labeled T4 of all tissues. Simultaneous injection of either 0.007 or 0.2 microgram T4 increased 3H-melatonin uptake into peripheral tissues that undergo major metamorphic changes but not into neural or endocrine organs. In contrast, 0.033, 3.75 or 15 micrograms melatonin had no significant influence on the content of 125I-T4 in any tissue studied in vivo. Results of in vitro labeling of selected tissues were generally in agreement with the in vivo work except that the 125I-T4 content of intestinal segments from late prometamorphic larvae was lower in melatonin-treated than in control groups. The results suggest that peripheral tissues are a major site for T4-melatonin interactions and that T4 may modulate its own action through influencing melatonin levels in target tissues and perhaps in the thyroid. Because melatonin had no effect on tissue T4 content in young tadpoles, retardation of metamorphic events by melatonin does not seem to involve modulation of T4 availability to the tissues.

Effect of melatonin on the response of the thyroid to thyrotropin stimulation in vitro.

Thyroidal-melatonin interactions are of particular importance to amphibian development since the thyroid controls the progress of metamorphosis while melatonin may coordinate its rate with prevailing environmental conditions. Melatonin antagonized thyroxine (T4) action at the tissue level and directly inhibited baseline T4 secretion in culture, so the present work sought to determine if it antagonized the response of the thyroid to thyroid stimulating hormone (TSH) as well. A preliminary experiment showed that, in Rana pipiens, the concentration of TSH (0.2 microg/ml) used in the culture of tadpole thyroids stimulated T4 secretion as much as frog pituitaries, but more than late premetamorphic tadpole pituitaries. There was no significant effect of 1 to 15 microg/ml melatonin in TSH-containing thyroid cultures of various Rana species of tadpoles and frogs in experiments with media collected once every 24 or 48 hr, although 15 microg/ml melatonin tended to depress T4 secretion. In a final experiment, a higher melatonin concentration was used as well as more frequent media collections. Thyroids from prometamorphic Rana catesbeiana tadpoles were cultured in L-15 media with periodic stimulation by 0.2 microg/ml TSH, or TSH and 10 or 100 microg/ml melatonin. Media were collected at the end of two 3-hr TSH pulses, and every 8 hr thereafter for the next 3 days. Melatonin was administered until the end of Day 2 while TSH was given only on Day 2 in addition to the original 3-hr pulses. The secretion of T4 was inhibited significantly by 10 microg/ml melatonin at only two of the early media collections. In contrast, 100 micro;g/ml melatonin significantly depressed T4 secretion in response to TSH at all but one interval and completely inhibited the thyroidal response to TSH reintroduced into the media on Day 2. The findings suggest that a high concentration of melatonin is inhibitory to the thyroidal response to TSH, but that lower concentrations do not significantly overcome the TSH stimulus.

A bitter struggle for integration survival.

The state of California created the El Camino Hospital District in 1961, one of 77 such districts created as a way to fund rural hospitals. During the 1990s, hospital administrators began to look for ways to streamline operations and cut costs to remain competitive. In 1992, the hospital District Board voted to turn over El Camino's management to a nonprofit company, believing the hospital could form business partnerships with doctors. The District created a private, nonprofit corporation, approved the appointment of volunteers and physicians to the board, and instructed the board to develop on alliance with other physicians. It became evident that several philosophies were at work regarding the healthcare system. In 1995, the District attempted to void the 1992 agreements and take back the hospital. Legal investigations were initiated. Meanwhile, reimbursement rates were decreasing and the hospital's 600 doctors were dissatisfied. Legal struggles continue, public controversy and physician anger are ongoing, and the Santa Clara County District Attorney's office has begun an audit of records. Camino Healthcare, despite all the ongoing difficulties, continues to provide excellent care to patients and the community.

Effect of melatonin on the anuran thyroid gland: follicle cell proliferation, morphometry, and subsequent thyroid hormone secretion in vitro after melatonin treatment in vivo.

The thyroid gland controls the progress of metamorphosis, although other hormones influence metamorphic rate, including melatonin, which may coordinate metamorphosis with seasonal and light conditions. Melatonin directly antagonized the action of thyroxine (T4) in promoting regression of tadpole tail tips in vitro, and this study sought to determine if it affects the thyroid axis of tadpoles as well. In an experiment sampling at 8-hr intervals for 24 hr, after melatonin treatment (15 micrograms/day for 12 days) of premetamorphic Rana pipiens tadpoles at approximately 1100 hr on 18L:6D, thyroid follicle cell height and lumen diameter were lowered by melatonin, but follicle cell proliferation was not significantly depressed. In a second experiment conducted under the same conditions, but sampling at 3-hr intervals for 24 hr, melatonin significantly lowered follicle cell labeling index and suppressed its ultradian (7.6 hr) rhythm, while shifting the peak of follicle lumen diameter to the dark instead of the light. Thus, melatonin tended to depress the thyroid of young tadpoles and suppress or shift its rhythms. Melatonin (10 micrograms/day for 5 days) injected into prometamorphic Rana catesbeiana tadpoles at 1930 hr on 18L:6D significantly altered subsequent in vitro thyroid function as determined by radioimmunoassay of media collected at intervals for 54 hr from cultured thyroids of injected control and melatonin groups, and a noninjected control group. Melatonin decreased T4 secretion during the first 30 hr, but not during the last 24 hr of culture, suppressed 3,5,3'-triidothyronine (T3) secretion for 12 hr, and then raised T3 output into the media above the control for the remainder of the culture period, increasing the T3:T4 ratio. Injection alone increased both T3 and T4 secretion for the first 30 hr, but did not change the T3:T4 ratio. The findings show that exogenous melatonin administered in vivo significantly modulated thyroid activity and morphometry directly and/or indirectly and comprise the first demonstration of an effect of melatonin on the amphibian thyroid gland.

Rotation and radical motion thresholds support a two-stage model of differential-motion analysis.

Lower motion thresholds for rotational and radial flow have been measured for stimuli consisting of four closely packed circular apertures, each containing patches of drifting grating or plaid. Detection and direction thresholds were measured for gratings and plaids as a function of the relative orientation of the pattern components. There was a similarity between both types of threshold, supporting the existence of specialised rotation and radial-flow detectors. Further, thresholds increased with the relative component orientation for both gratings and plaids. This suggests that component information from a first stage, tuned spatiotemporally and to orientation, is being used directly to compute the optic flow in a two-stage process. A model based on this architecture is described by means of simple template receptive-field arrays with separable temporal and spatial tuning at the first stage.

Circadian rhythms of thyroid secretion, morphometry, and cell division in prometamorphic and climax Rana tadpoles.

Rhythmicity of thyroid follicle structure, cell division, and T4 secretion in vitro was studied in anuran tadpoles on a 12L:12D cycle with light onset at 0800 hr. Stage XIII Rana pipiens larvae had a significant circadian rhythm of follicle lumen diameter (acrophase 1052 hr). Follicle cell height was higher at 0900 hr than at other times but the rhythm was not significant. At Stage XX, both follicle cell height (acrophase 2318 hr) and cell division (acrophase 1929 hr) were rhythmic while lumen diameter, though highest at 0900 hr, showed no significant daily changes. Thyroids from Rana catesbeiana larvae at Stages XVII to XVIII had a significant circadian rhythm of T4 secretion (acrophase 2034 hr) in vitro as measured by radioimmunoassay of media collected every 6 hr for 24 hr. The findings indicate that the daily sequence of thyroid structure and function in preclimax Rana tadpoles includes larger follicle lumina early in the light and maximum cell division, T4 secretion, and follicle cell height within a 4-hr interval beginning around the onset of dark.

Anterior pituitary and adrenal cortical hormones accelerate or inhibit tadpole hindlimb growth and development depending on stage of spontaneous development or thyroxine concentration in induced metamorphosis.

The effect of prolactin, growth hormone, and various adrenal corticoids on hindlimb growth, development, and differentiation was studied in Rana pipiens larvae. Experiments were performed at different stages of spontaneous development and during metamorphosis induced in premetamorphic tadpoles by various concentrations of exogenous T4. Prolactin at 10 micrograms/day inhibited the limb at spontaneous premetamorphosis, had no effect at prometamorphosis or when administered with 3.8 nM T4, and synergized with T4 at 63 nM T4 and above. Growth hormone (10 or 20 micrograms/day) promoted limb growth and development during premetamorphosis but had no effect on spontaneous or induced metamorphosis thereafter, nor did it stimulate limb epidermal differentiation. The adrenal corticoids inhibited limb growth and epidermal cell proliferation during pre- and prometamorphosis but had no effect on limb morphogenesis or differentiation. The depressive effect of corticoids during spontaneous metamorphosis is at least partly through thyroid inhibition since hydrocortisone significantly reduced follicle cell height, lumen diameter, and cell proliferation in the thyroid. During induced metamorphosis, steroids (0.29 microM), especially corticosterone and aldosterone, antagonized the effect of 0.38 to 1.2 nM T4 on the limb. All steroids except deoxycorticosterone synergized with 3.8 nM T4, and at 31 nM T4, approximating the climax level with permeability factors taken into account, all corticoids synergized with T4 to promote limb growth and development. Aldosterone antagonized T4 at a higher T4 level than the other corticoids. The effect of all steroids except corticosterone was also corticoid dose-dependent. The results show the importance of the T4 concentrations in interactions of T4 with other hormones and suggest a scheme for hormonal control of limb growth and morphogenesis during metamorphosis. During premetamorphosis growth hormone synergizes with low endogenous T4 to promote initial limb growth and development while prolactin opposes this action. During prometamorphosis, as growth hormone and prolactin become ineffective corticosteroids begin to synergize with the rising level of endogenous T4. At climax, prolactin also augments the action of T4 to bring about rapid hindlimb growth.

DNA synthesis is unaffected but subsequent cell division is delayed in tadpole hindlimb epidermis when thyroxine is given in the dark.

In Rana pipiens tadpoles, thyroxine (T4) injection in the early light promotes faster hindlimb growth and development than injection early in the dark. T4 also stimulates a significant increase in cell proliferation in the basal epidermal cells of the limb. Using autoradiography, we studied the timing of the T4-induced rise in the labeling and mitotic indices on 12L:12D cycles with 0.2 micrograms T4 injection early in the light or dark and on a 12L:3D:1L:8D cycle with a light pulse early in the dark shortly after a T4 injection. In all instances, the labeling index peaked at the same time after the start of T4, so diurnal differences in the binding or initial actions of T4 leading up to the entrance of epidermal cells into the DNA synthetic (S) phase of the cell cycle are not indicated. However, with T4 injection in the dark the subsequent mitotic index peak was delayed, to a greater extent on 12L:12D than on 12L:3D:1L:8D. Cells induced to proliferate following T4 injection in the dark evidently had longer cell cycles, probably at the expense of the S or G2 phase, than when T4 was given in the light.

Metamorphic rate as a function of the light/dark cycle in Rana pipiens larvae.

1. The rate of development of Rana pipiens tadpoles in spontaneous and thyroxine (T4)-induced metamorphosis was studied on light/dark (LD) cycles in which the photophase was held constant while the scotophase was progressively extended or vice versa. 2. Metamorphic rate fluctuated in both types of experiments as the LD cycle lengthened. However, the pattern of resonance differed with the length of the photophase. For example, with an 8 hr light phase, development rate slowed and then increased as the cycle was extended from 24 to to 36 hr, whereas with a 12 hr photophase the reverse took place. 3. The findings are compatible with the occurrence of a rhythm of light sensitivity in photoperiodic time measurement in this amphibian. 4. From the viewpoint of hormonal mechanisms, it is suggested that photoperiodic effects on metamorphic rate result from different patterns of melatonin secretion under the various LD cycles, since melatonin can modify the action of T4 in metamorphosis.