Bio-scaffolds in organ-regeneration: Clinical potential and current challenges.

Cadaveric organ transplantation represents the definitive treatment option for end-stage disease but is restricted by the shortage of clinically-viable donor organs. This limitation has, in part, driven current research efforts for in vitro generation of transplantable tissue surrogates. Recent advances in organ reconstruction have been facilitated by the re-purposing of decellularized whole organs to serve as three-dimensional bio-scaffolds. Notably, studies in rodents indicate that such scaffolds retain native extracellular matrix components that provide appropriate biochemical, mechanical and physical stimuli for successful tissue/organ reconstruction. As such, they support the migration, adhesion and differentiation of reseeded primary and/or pluripotent cell populations, which mature and achieve functionality through short-term conditioning within specialized tissue bioreactors. Whilst these findings are encouraging, significant challenges remain to up-scale the present technology to accommodate human-sized organs and thereby further the translation of this approach towards clinical use. Of note, the diverse structural and cellular composition of large mammalian organ systems mean that a "one-size fits all" approach cannot be adopted either to the methods used for their decellularization or the cells required for subsequent re-population, to create fully functional entities. The present review seeks to highlight the clinical potential of decellularized organ bio-scaffolds as a route to further advance the field of tissue- and organ-regeneration, and to discuss the challenges which are yet to be addressed if such a technology is ever to become a credible rival to conventional organ allo-transplantation.

Modeling emissions from CAFO poultry farms in Poland and evaluating potential risk to surrounding populations.

The world-wide use of concentrated animal feeding operations (CAFOs) for livestock production demands the need to evaluate the potential impact to public health. We estimated the exposure of various airborne pollutants for populations residing in close proximity to 10 poultry CAFOs located in Central Poland. Ammonia (NH3), carbon dioxide (CO2), carbon monoxide (CO), hydrogen sulfide (H2S), methane (CH4), nitrogen dioxide (NO2), nitrous oxide (N2O), sulfur dioxide (SO2), and organic dust were the pollutants of interest for this study. Because no monitoring data were available, we used the steady-state Gaussian dispersion model AERMOD to estimate pollutant concentrations for the exposed population in order to calculate the hazard index (HI) for a combined mixture of chemicals. Our results indicate that while the levels of certain pollutants are expected to exceed background levels commonly found in the environment they did not result in calculated hazard indexes which exceeded unity suggesting low potential for adverse health effects for the surrounding community for the mixture of chemicals. The study was conducted through a cooperation between the Agency for Toxic Substances and Disease Registry (ATSDR) in the USA and the Nofer Institute of Occupational Medicine (NIOM) in Poland.

Public health decisions: actions and consequences.

The goal of public health is to promote the best possible health for the whole population. Public health issues are numerous and can be unbelievably complex in form, scope, and possible consequence. Most public health decisions involve assessing several different options, weighing the respective benefits and risks of those options, and making difficult decisions that hopefully provide the greatest benefit to the affected populations. Many risk management decisions involve a variety of societal factors which modify risk assessment choices. The purpose of this paper is to point out difficulties in making decisions that impact public health. The intent of such decisions is to improve public health, but as illustrated in the paper, there can be unintended adverse consequences. Such unplanned issues require continued attention and efforts for responsible officials in the protection of environmental public health. This article presents examples of such events, when in the past, it was necessary to assess and regulate a number of potentially hazardous chemicals commonly used as insecticides, gasoline additives, and wood preservatives.

Rotational co-culture of clonal ß-cells with endothelial cells: effect of PPAR-γ agonism in vitro on insulin and VEGF secretion.

AIM: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. METHODS: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-γ agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. RESULTS: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p < 0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p < 0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p < 0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p < 0.01) and VEGF production (p < 0.01) in the presence and absence of D11 cells. CONCLUSIONS: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-γ agonist may improve the prospects for graft revascularization and function after implantation.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system.

AIMS/HYPOTHESIS: Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. METHODS: Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. RESULTS: Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p < 0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. CONCLUSIONS/INTERPRETATION: The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Dose- and sex-dependent effects of the neurotoxin 6-hydroxydopamine on the nigrostriatal dopaminergic pathway of adult rats: differential actions of estrogen in males and females.

Epidemiological and clinical studies provide growing evidence for marked sex differences in the incidence of certain neurological disorders that are largely attributed to the neuroprotective effects of estrogen. Thus there is a keen interest in the clinical potential of estrogen-related compounds to act as novel therapeutic agents in conditions of neuronal injury and neurodegeneration such as Parkinson's disease. Studies employing animal models of neurodegeneration in ovariectomised female rats treated with estrogen support this hypothesis, yet experimental evidence for sex differences in the CNS response to direct neurotoxic insult is limited and, as yet, few studies have addressed the role played by endogenously produced hormones in neuroprotection. Therefore, in this study we aimed to determine (1) whether the prevailing levels of sex steroid hormones in the intact rat provide a degree of protection against neuronal assault in females compared with males and (2) whether sex differences depend solely on male/female differences in circulating estrogen levels or whether androgens could also play a role. Using the selective, centrally administered neurotoxin 6-hydroxydopamine, which induces a lesion in the nigrostriatal dopaminergic pathway similar to that seen in Parkinson's disease, we have demonstrated a sexually dimorphic (male-dominant), dose-dependent susceptibility in rats. Furthermore, following gonadectomy, dopamine depletion resulting from a submaximal dose of 6-hydroxydopamine (1 microg) was reduced in male rats, whereas in females, ovariectomy enhanced dopamine depletion. Administration of the nonaromatizable androgen dihydrotestosterone to gonadectomized animals had no significant effect on 6-hydroxydopamine toxicity in either males or females, whereas treatment of gonadectomized males and females with physiological levels of estrogen restored the extent of striatal dopamine loss to that seen in intact rats, viz, estrogen therapy reduced lesion size in females but increased it in males. Taken together, our findings strongly suggest that there are sex differences in the mechanisms whereby nigrostriatal dopaminergic neurones respond to injury. They also reveal that the reported clinically beneficial effects of estrogen in females may not be universally adopted for males. While the reasons for this gender-determined difference in response to the activational action of estrogen are unknown, we hypothesize that they may well be related to the early organizational events mediated by sex steroid hormones, which ultimately result in the sexual differentiation of the brain.

Randomized placebo-controlled trial of metronidazole 1% cream with sunscreen SPF 15 in treatment of rosacea.

BACKGROUND: Rosacea is a photoaggravated dermatosis responsive to treatment with topical and oral antibiotics. A formulation combining metronidazole 1% cream with sunscreen SPF 15 was developed for the treatment of rosacea. OBJECTIVE: The objective of this study was to determine the safety and efficacy of a formulation combining metronidazole 1% cream with sunscreen SPF 15 in the treatment of moderate to severe rosacea. METHODS: One hundred and twenty patients with moderate to severe rosacea were enrolled for a randomized, placebo-controlled (vehicle containing sunscreen with SPF 15), double-blind study. Study cream was applied twice daily to the entire face over a 12-week period. RESULTS: Treatment with metronidazole 1% cream with sunscreen SPF 15 resulted in significant improvement (p <0.05) in inflammatory lesion count, erythema and telangiectasiae scores, and investigator and patient global assessment scores compared with baseline and placebo. Adverse reactions related to study medication were typically mild, occurred at the site of application, and were reversible. There was no difference between the safety profiles of metronidazole 1% cream with sunscreen SPF 15 and placebo. CONCLUSIONS: The combined topical formulation of metronidazole 1% cream with sunscreen SPF 15 was an effective, well-tolerated topical agent for the treatment of moderate to severe rosacea.

Correlation of hypothalamic somatostatin mRNA expression and peptide content with secretion: sexual dimorphism and differential regulation by gonadal factors.

Sex differences in growth hormone (GH) secretion in the rat are thought to be determined, to a large extent, by gonadal steroid-dependent sex differences in somatostatin (SRIH) secretion from neurones in the periventricular nucleus (PeN) which project to the median eminence (ME). The present study aimed to obtain direct evidence for sex differences and gonadal regulation of SRIH release within this pathway and to determine the relationships between SRIH mRNA expression, SRIH peptide content and SRIH secretion in the adult rat. Somatostatin mRNA expression in the PeN and peptide content in both PeN and ME were higher in males than females (P<0.05). However, both basal and 56 mM K+-stimulated SRIH release in vitro from hypothalamic explants incorporating the PeN-ME pathway were higher (P<0.01) in females. The gonadectomy of female rats resulted in significantly reduced basal levels of SRIH release equivalent to that of males but had no effect on SRIH mRNA/peptide content or K+-stimulated release. In contrast, gonadectomy of male rats reduced SRIH mRNA and peptide contents and elevated K+-stimulated secretion (P<0.01) to levels similar to that seen in intact females, without affecting basal release. In summary, these results demonstrate that in the PeN-ME of the adult rat: (1) SRIH mRNA and peptide content is well correlated and sexually dimorphic but dependent on gonadal factors in the male only; (2) SRIH secretion is sexually dimorphic and dependent on gonadal factors; but (3) differences in mRNA/peptide content do not reflect secretory capacity; and (4) gonadal factors differentially modulate SRIH secretory dynamics in males and females.

Ontogeny and sexual differentiation of somatostatin biosynthesis and secretion in the hypothalamic periventricular-median eminence pathway.

The biosynthesis of somatostatin (SRIH) in the hypothalamic periventricular nucleus (PeN) is sexually differentiated in neonatal and adult rats by virtue of the organizational and activational actions, respectively, of sex steroid hormones. Little information exists, however, on the normal pattern of maturation of these neurones or on how the sexually differentiated biosynthesis may relate to ontogenetic changes in somatostatin secretion during the neonatal and pubertal periods of development. Hence in the present study we determined the postnatal developmental profile of SRIH mRNA and peptide levels in the PeN-median eminence (ME) pathway as well as SRIH secretion, using an acute explant preparation, from the day of birth, through puberty and into adulthood in male and female rats. The results demonstrate that: (1) developmental sex differences in SRIH biosynthesis in PeN neurones occurred in an orderly cascade with differences observed for mRNA expression at postnatal day 5, for peptide content in the perikarya at postnatal day 10 and for peptide content in the nerve terminal (ME) by postnatal day 25; (2) sex differences in SRIH release were not evident prior to postnatal day 40; and (3) the developmental profile of SRIH biosynthesis in PeN neurones is unique compared with other hypothalamic (ventromedial nucleus) and extrahypothalamic (parietal cortex) populations. Specific developmental changes in the biosynthetic and secretory activity of the hypothalamic SRIH PeN-ME pathway may have a functional importance in the maturation of hypothalamic SRIH pathways involved in the regulation of GH secretion.

Sexually dimorphic ontogeny of GABAergic influences on periventricular somatostatin neurons.

The biosynthesis and secretion of somatostatin (SRIH) within the hypothalamic periventricular-median eminence (PeN-ME) pathway follows a sexually differentiated developmental pattern beginning in the early neonatal period. It is generally accepted that testosterone plays a role in these processes, but the mechanisms underlying the age and sex differences are poorly understood. The present study sought to investigate the hypothesis that gamma-aminobutyric acid (GABA) may play a role in determining sex differences in SRIH neuronal activity. Using an in vitro hypothalamic preparation where more than 97% of the immunoreactive SRIH is contained within the PeN-ME pathway, peptide release in response to the GABA(A) receptor antagonist, bicuculline, was followed through development. In the male a stimulatory response, indicative of an inhibitory GABAergic tone on SRIH secretion, was observed as early as postnatal day (P) 5. This persisted throughout juvenile development (P10, P17) and was present also in the adult male (P75), but in the peripubertal period the response to bicuculline was first lost (P25) and then reversed to an inhibition (P40), suggesting a transient switch to an apparent stimulatory GABAergic tone on SRIH release. By contrast, in the female, no bicuculline responsiveness was seen until P25 when it caused a decrease in SRIH release which persisted into adulthood. Using in situ hybridization studies we found no evidence to support the view that these age- and sex-dependent differences were due to changes in the expression of GABA(A) receptor alpha-subunits (alpha(1) and alpha(2)) which are colocalised in the PeN SRIH neurons. Following adult gonadectomy, the bicuculline response was abolished in the male, whereas, in the female it was reversed and identical in magnitude to the response in the intact male. These results demonstrate marked sex differences in GABA(A)-receptor-mediated influences on SRIH release which develop soon after birth and, in the adult, depend on gonadal factors. In the male these factors activate a primarily inhibitory influence, whereas in the female they facilitate an apparently stimulatory tone of GABA on SRIH secretion via the GABA(A) receptor. Our findings thus support the view that GABAergic transmission may play a key role in generating sex differences in the mode of SRIH secretion from the hypothalamus which has been shown to be a major factor in determining the sexually dimorphic patterns of growth hormone secretion.

The use of hematological effects in the development of minimal risk levels.

The Agency for Toxic Substances and Disease Registry (ATSDR) derives minimal risk levels (MRLs) to assist in evaluating risk of adverse health effects in individuals exposed to hazardous substances. MRLs are derived from published values identifying no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in animal or human studies. The most sensitive end points are used. To date, 4 inhalation MRLs and 13 oral MRLs have been derived from hematological end points for 12 substances. This paper provides a brief overview of the hematological system, examples of hematological end points, and the MRL for substances with hematological end points.

Estrogen-dependent ontogeny of sex differences in somatostatin neurons of the hypothalamic periventricular nucleus.

The sexually dimorphic profile of GH secretion is thought to be engendered by gonadal steroids acting in part on hypothalamic periventricular somatostatin (SOM) neurons. The present study set out to examine and characterize the development of sex differences in these SOM neurons. In the first series of experiments, we used in situ hybridization to examine SOM messenger RNA (mRNA) expression within the periventricular nucleus (PeN) of male and female rats on postnatal day 1 (P1), P5, and P10. Cellular SOM mRNA content was found to increase from P1 to P10 in both sexes (P < 0.01), but was 24% (P < 0.05) and 38% (P < 0.01) higher in males on P5 and P10, respectively. A second series of experiments examined the SOM peptide content of the PeN in developing rats and found increasing levels from P1 to P10, with a 44% higher SOM content in males compared with females on P10 (P < 0.05). The third series of experiments questioned the role of gonadal steroids in engendering sex differences in SOM mRNA expression by determining the effects of neonatal gonadectomy (GDX) and replacement of dihydrotestosterone or estradiol benzoate. The SOM mRNA content of PeN neurons in P5 males gonadectomized on the day of birth was the same as that in P5 females and was significantly reduced compared with that in sham-operated P5 males (P < 0.05). Male rats GDX on P1 and treated with estradiol benzoate from P1 to P5 had cellular SOM mRNA levels similar to those in intact males on P5, whereas dihydrotestosterone treatment had no effect. Treatment of intact males with an androgen receptor antagonist, cyproterone acetate, on P1 had no effect on cellular SOM mRNA on P5, whereas male rats given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione from P1 to P5 had lower (P < 0.05) SOM mRNA levels than controls. In the final set of experiments, dual labeling immunocytochemistry showed that SOM neurons in the PeN of P5 rats did not contain estrogen receptor-alpha, but expressed androgen receptors in a sexually dimorphic manner. These results demonstrate that a sex difference in SOM biosynthesis, which persists into adulthood, develops between P1 and P5 in PeN neurons. Despite the absence of estrogen receptor-alpha in these neurons, the organizational influence of testosterone only occurs after its aromatization to estrogen.

Differential effects of neuroactive steroids on somatostatin and dopamine secretion from primary hypothalamic cell cultures.

This study investigated the effects of neuroactive steroids, which have been reported to modulate GABA-ergic transmission, on the secretion of somatostatin (SRIH) and also dopamine (DA) from primary rat hypothalamic cell cultures, where the release of both substances is regulated by a GABAA receptor-mediated inhibitory tone. Pregnenolone sulphate (PS), a negative allosteric modulator at the GABAA receptor, enhanced SRIH secretion in a time and dose-dependent manner (10(-12)-10(-8) M). This effect was reversed by muscimol (10(-8) M) and enhanced by bicuculline (10(-6) M), thus supporting an action of PS at the GABAA receptor. The release of endogenously synthesized dopamine (DA) was, however, unaffected by PS. A number of other steroids were also tested for their potential actions on SRIH and DA secretion. Allopregnanolone had slight but significant stimulatory actions on SRIH secretion, whereas tetrahydro-deoxycorticosterone (TH-DOC) markedly stimulated SRIH secretion with a bell-shaped dose response curve resembling that found for PS. The release of DA was unaffected by these neuroactive steroids but, unlike SRIH, DA release was stimulated by dehydroepiandrosterone sulphate (DHEAS). The results support the view that neuroactive steroids may play an important role in regulating some aspects of neuroendocrine function and they also provide the first demonstration of differential activities of neuroactive steroids within the hypothalamus at low, physiologically relevant concentrations. The results also raise the possibility that certain hypothalamic neuronal populations may possess uniquely different GABAA receptors and that such mechanisms may contribute to the functional development of the neuroendocrine system.

Quantitation of H1-receptor antagonists in skin and serum.

BACKGROUND: Cetirizine and hydroxyzine produce prompt, long-lasting peripheral H1-blockade in skin. We hypothesized that after oral administration of these H1-receptor antagonists, skin concentrations would be higher than serum concentrations and would correlate with peripheral H1 blockade. METHODS: In a randomized, double-blind, parallel-group study in 13 healthy subjects, skin biopsies, venipunctures, and epicutaneous tests with histamine were performed before ingestion of 10 mg cetirizine or 50 mg hydroxyzine and 1, 3, 6, 9, and 24 hours after administration. Subjects then took 10 mg cetirizine or 50 mg hydroxyzine at 21:00 hours for 6 consecutive days. All tests were repeated at 168 hours (steady state), 12 hours after the last dose. RESULTS: Skin cetirizine concentrations were lower than serum ccetirizine concentrations from 1 to 9 hours but were higher at 24 hours and equivalent at 168 hours (steady state). Skin hydroxyzine concentrations were higher than serum hydroxyzine concentrations at all test times. After hydroxyzine dosing, cetirizine, the active metabolite of hydroxyzine arising in vivo, was found in skin and serum. Single doses of cetirizine or hydroxyzine produced highly significant suppression of wheals and flares from 3 to 24 hours inclusive, and this suppression was maintained at steady state. CONCLUSIONS: Cetirizine and hydroxyzine enter the skin readily, and their sustained high concentrations in skin after single or multiple dosing may contribute to their well-known efficacy in symptomatic treatment of urticaria and other skin disorders in which histamine plays a role.

Investigation of the ontogenetic patterns of rat hypothalamic dopaminergic neurone morphology and function in vitro.

Using fetal rat hypothalamic cells in primary culture maintained in a serum-free defined medium we have investigated the morphological and functional development of the dopamine (DA)-containing neurones intrinsic to the hypothalamus. Immunocytochemical studies demonstrated the presence of three morphologically distinct subtypes of tyrosine hydroxylase-immunopositive neurones. On day 3 in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two subtypes persisted to later days in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo. Biochemical studies employing [3H]DA demonstrated a time- and temperature-dependent uptake mechanism within the cultures which was significantly attenuated by the uptake inhibitors benztropine and nomifensine in a dose-dependent manner. [3H]DA was also released under both basal and 56 mmol K+/l-stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The amount of [3H]DA accumulated and released by the cultural cells increased with the age of the culture, suggesting functional maturation of the DA-containing neurones within this preparation. The role of oestradiol-17 beta in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of [3H]DA and by direct measurement of endogenously synthesized DA using high-performance liquid chromatography coupled with electrochemical detection. Both uptake and release of [3H] and release of endogenous DA were significantly modulated by the concentration of steroid in the defined medium. These results demonstrate that hypothalamic dopaminergic neurones, when maintained in primary culture, undergo morphological and functional maturation which have several correlates in vivo. In addition, we have demonstrated that at least one sub-population of dopaminergic neurones within this preparation is responsive to oestradiol-17 beta. As DA is considered to be a vital component in the regulation of neuroendocrine activity we suggest that this model is valuable for the investigation of the functional development of the DA systems of the hypothalamus and the relationship existing between neurotransmitters, neuropeptides and neuroactive steroids.