Social Environment Ameliorates Behavioral and Immune Impairments in Tyrosine Hydroxylase Haploinsufficient Female Mice.

The social environment can influence the functional capacity of nervous and immune systems, and consequently the state of health, especially in aged individuals. Adult female tyrosine hydroxylase haploinsufficient (TH-HZ) mice exhibit behavioral impairments, premature immunosenescence and oxidative- inflammatory stress. All these deteriorations are associated with a lower lifespan than wild type (WT) counterparts. The aim was to analyze whether the cohabitation with WT animals could revert or at least ameliorate the deterioration in the nervous and immune systems that female TH-HZ mice show at adult age. Female TH-HZ and WT mice at age of 3-4 weeks were divided into following groups: control TH-HZ (5 TH-HZ mice in the cage; TH-HZ100%), control WT (5 WT mice in the cage; WT100%), TH-HZ > 50% and WT < 50% (5 TH-HZ with 2 WT mice in each cage) as well as TH-HZ < 50% and WT > 50% (2 TH-HZ and 5 WT mice in each cage). At the age of 37-38 weeks, all mice were submitted to a battery of behavioral tests, evaluating sensorimotor abilities, exploratory capacities and anxiety-like behaviors. Subsequently, peritoneal leukocytes were extracted and several immune functions as well as oxidative and inflammatory stress parameters were analyzed. The results showed that the TH-HZ < 50% group had improved behavioral responses, especially anxiety-like behaviors, and the immunosenescence and oxidative stress of their peritoneal leukocytes were ameliorated. However, WT mice that cohabited with TH-HZ mice presented higher anxiety-like behaviors and deterioration in immune functions and in their inflammatory stress parameters. Thus, this social environment is capable of ameliorating the impairments associated with a haploinsufficiency of the th gene. Graphical Abstract.

Red blood cell distribution width [RDW] and long-term mortality after community-acquired pneumonia. A comparison with proadrenomedullin.

Proadrenomedullin (proADM), a cardiovascular biomarker, has shown high prognostic power for community-acquired pneumonia (CAP) outcomes. Red-blood-cell distribution width (RDW), linked to cardiovascular disorders, has been associated with short-term and medium-term mortality after CAP. Our objective was to assess the accuracy of both biomarkers for CAP long-term mortality (>90 days). Adults hospitalized with CAP underwent blood proADM, RDW, C-reactive protein (CRP) and procalcitonin (PCT) measurements at admission, and were evaluated after 30, 90, and 180 days, and 1, 2, and 3 years, until either death or 5 years of follow-up. A group of 265 patients were recruited, with an average follow-up 1018 ± 539 days. Of these, 217 were followed for 1 year, and 187 for 3 years. Levels of both proADM and RDW were higher in those who died in the short term (p = 0.017 and p < 0.0001, respectively), medium term (p = 0.004 and p < 0.0001, respectively) and long term (p < 0.0001 and p < 0.0001, respectively). RDW showed lower accuracy (30-day AUC, 0.673) than proADM (AUC, 0.816), PSI (AUC, 0.846), and CURB65 (AUC, 0.817) scores for short-term and medium-term mortality prediction. However, accuracy was similar (3-year AUC, 0.692, 0.698, 0.743, and 0.704, respectively) for long-term mortality, and RDW > 14% (RDW > 14) increased the prediction power of both PSI (AUC, 0.743 vs 0.779; p < 0.0001) and CURB65 (AUC, 0.704 vs 0.747; p < 0.0001) scores, as did proADM. RDW > 14 + PSI and RDW > 14 + CURB65 associations had a sensitivity for long-term mortality of 80.8%-90% and 74%-90%, and a specificity of 56.7%-61.5% and 59.3%-64.2%, respectively. Both proADM and RDW > 14 (HR, 4.116) were independent risk factors for long-term mortality and were associated with poorer survival. Our findings agree with the suggested association between cardiovascular disease and long-term CAP mortality. RDW, routinely provided as part of the whole blood count, and especially associated with clinical scores, can provide useful information about long-term CAP outcomes.

Proinsulin in development: New roles for an ancient prohormone.

In postnatal organisms, insulin is well known as an essential anabolic hormone responsible for maintaining glucose homeostasis. Its biosynthesis by the pancreatic beta cell has been considered a model of tissue-specific gene expression. However, proinsulin mRNA and protein have been found in embryonic stages before the formation of the pancreatic primordium, and later, in extrapancreatic tissues including the nervous system. Phylogenetic studies have also confirmed that production of insulin-like peptides antecedes the morphogenesis of a pancreas, and that these peptides contribute to normal development. In recent years, other roles for insulin distinct from its metabolic function have emerged also in vertebrates. During embryonic development, insulin acts as a survival factor and is involved in early morphogenesis. These findings are consistent with the observation that, at these stages, the proinsulin gene product remains as the precursor form, proinsulin. Independent of its low metabolic activity, proinsulin stimulates proliferation in developing neuroretina, as well as cell survival and cardiogenesis in early embryos. Insulin/proinsulin levels are finely regulated during development, since an excess of the protein interferes with correct morphogenesis and is deleterious for the embryo. This fine-tuned regulation is achieved by the expression of alternative embryonic proinsulin transcripts that have diminished translational activity.

Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.

Transforming growth factor (TGF)-beta and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-beta and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-beta resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-beta-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-beta receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-beta signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-beta activity.

Insulin-like growth factor 1 is required for survival of transit-amplifying neuroblasts and differentiation of otic neurons.

Neurons that connect mechanosensory hair cell receptors to the central nervous system derive from the otic vesicle from where otic neuroblasts delaminate and form the cochleovestibular ganglion (CVG). Local signals interact to promote this process, which is autonomous and intrinsic to the otic vesicle. We have studied the expression and activity of insulin-like growth factor-1 (IGF-1) during the formation of the chick CVG, focusing attention on its role in neurogenesis. IGF-1 and its receptor (IGFR) were detected at the mRNA and protein levels in the otic epithelium and the CVG. The function of IGF-1 was explored in explants of otic vesicle by assessing the formation of the CVG in the presence of anti-IGF-1 antibodies or the receptor competitive antagonist JB1. Interference with IGF-1 activity inhibited CVG formation in growth factor-free media, revealing that endogenous IGF-1 activity is essential for ganglion generation. Analysis of cell proliferation cell death, and expression of the early neuronal antigens Tuj-1, Islet-1/2, and G4 indicated that IGF-1 was required for survival, proliferation, and differentiation of an actively expanding population of otic neuroblasts. IGF-1 blockade, however, did not affect NeuroD within the otic epithelium. Experiments carried out on isolated CVG showed that exogenous IGF-1 induced cell proliferation, neurite outgrowth, and G4 expression. These effects of IGF-1 were blocked by JB1. These findings suggest that IGF-1 is essential for neurogenesis by allowing the expansion of a transit-amplifying neuroblast population and its differentiation into postmitotic neurons. IGF-1 is one of the signals underlying autonomous development of the otic vesicle.

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P(0) in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Mexican Americans with Type 2 diabetes mellitus.

BACKGROUND: Insulin receptor substrate-1 (IRS-1) is an endogenous substrate for the insulin receptor tyrosine kinase, which plays an important role in insulin signaling. Mutations in the IRS-1 gene are associated in some populations with obesity and Type 2 diabetes. METHODS: To determine whether variation in the IRS-1 gene contributes to genetic susceptibility to insulin resistance and Type 2 diabetes in Mexican Americans, the entire coding region of the IRS-1 gene was screened for variation in 31 unrelated subjects with Type 2 diabetes using single-stranded conformational polymorphism analysis (SSCP) and dideoxy sequence analysis. Variants encoding amino acid substitutions were genotyped in 27 unrelated nondiabetic Mexican Americans and in all family members of subjects containing these variants, and association analyses were performed. To trace the ancestral origins of the variants, Iberian Caucasians and Pima Indians were also genotyped. RESULTS: Eight single base changes were found: four silent polymorphisms and four missense mutations (Ala94Thr, Ala512Pro, Ser892Gly and Gly971Arg). Allele frequencies were 0.009, 0.017, 0.017 and 0.043, respectively. There were no significant associations of any of these variants with diabetes, glucose or insulin levels during an oral glucose tolerance test, or with body mass index (BMI) in Mexican American families except for a modest association between the Ala94Thr variant and decreased BMI (30.4 kg/m(2) vs 24.0 kg/m(2); p=0.035). None of these four missense mutations were detected in Pima Indians. In Iberian Caucasians, neither Ala94Thr nor Ser892Gly were detected, and Ala512Pro was detected in only 0/60 diabetic patients and 1/60 nondiabetic controls. Gly971Arg was relatively more common in Iberian Caucasians with 12/58 diabetic patients and 7/60 nondiabetic controls being heterozygous for this variant (p=0.21 for comparison between diabetic and nondiabetic subjects). CONCLUSIONS: Ala94Thr, Ala512Pro and Ser892Gly mutation are rare in the populations studied. Gly971Arg, is more common in Mexican Americans and Caucasians, but is not a major contributor to genetic susceptibility to Type 2 diabetes.

Cell death in early neural development: beyond the neurotrophic theory.

The important effect of cell death on projecting neurons during development is well established. However, this mainstream research might have diverted recognition of the cell death that occurs at earlier stages of neural development, affecting proliferating neural precursor cells and young neuroblasts. In this article, we briefly present observations supporting the occurrence of programmed cell death during early neural development in a regulated fashion that to some extent parallels the death of projecting neurons lacking neurotrophic support. These findings raise new questions, in particular the magnitude and the role of this early neural cell death.

Spatial and temporal variability of ground-level ozone in Castilla-León (Spain).

Ozone is a secondary air pollutant that has received extensive attention in the literature, mainly because of the adverse effects that exposure to it can cause, particularly in vegetation during the growing season. Because meteorological conditions strongly influence the efficiency of photochemical processes leading to ozone formation and destruction, ground-level ozone air pollution is currently being considered as a regional-scale phenomenon rather than a local one. This role of O3 as a regional-scale pollutant often implies the handling of large data sets in order to obtain information about its spatial and temporal variability patterns over a given broad region. Rotated principal component analysis (RPCA) is known to be one of the most powerful mathematical tools that can be used to achieve this aim. RPCA was applied in this paper to the summer and winter hourly time series of ground-level O3, concentrations recorded during 2 consecutive years (1996-1997) at 26 urban and suburban sites in Castilla-León (Spain). This procedure permitted the identification of different subregions where O3 concentrations show different spatio-temporal variability patterns. These variability patterns are mainly associated with the interaction of regional-level meteorological and anthropogenic factors. Some differences between winter and summer patterns were also found.

IGF-I expression is decreased in LIF-deficient mice after peripheral nerve injury.

We investigated the regulation of insulin-like growth factor 1 (IGF-1) expression after sciatic nerve crush using leukemia inhibitory factor (LIF)-deficient mice. One day post-crush, IGF-1 mRNA levels were lower in the LIF-deficient mouse nerve than in the wild type nerve. IGF-1 protein, analyzed by immunohistochemistry, was also decreased 1 day post-crush in LIF-deficient nerves relative to wild type nerves. By 3 days post-crush, IGF-1 immunoreactivity was induced in Schwann cells to equivalent levels in both types of nerve. After crush, IGF-1 expression was also found in mast cells, and these were initially decreased in the LIF-deficient mice. Thus, LIF appears to regulate IGF-1 expression in the peripheral nerve basally and early in the regeneration response in vivo.

c-Raf regulates cell survival and retinal ganglion cell morphogenesis during neurogenesis.

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.

In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis.

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Díaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624-1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.

Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro.

The cytokine leukaemia inhibitory factor (LIF) is up-regulated in glial cells after injury to the peripheral and central nervous systems. In addition, LIF is required for the changes in neuropeptide expression that normally occur when the axons of sympathetic and sensory neurons are transected. We investigated whether LIF is also necessary for the initial inflammatory response that follows mechanical injury to the sciatic nerve and cerebral cortex of adult mice. We find that inflammatory cell infiltration into crushed sciatic nerve is significantly slower in LIF knock-out (KO) mice compared with wild-type (WT) mice. Similarly, the microglial and astroglial responses to surgical injury of the cortex are significantly slower in LIF KO mice compared with WT mice. Consistent with these in vivo results, LIF is chemotactic for peritoneal macrophages in a microchamber culture assay. Thus, LIF is a key regulator of neural injury in vivo, where it is produced by glia and can act directly on neurons, glia and inflammatory cells. We also find that the initial inflammatory response to cortical injury is diminished in interleukin (IL)-6 KO mice. Surprisingly, however, the inflammatory response in LIF-IL-6 double KO mice is very similar to that of the single KO mice, suggesting that these cytokines may act in series rather than in parallel in this response.

Dynamic restricted expression of the chaperone Hsc70 in early chick development.

The non-inducible chaperone heat shock cognate 70 kDa (Hsc70) is regulated during development. We now characterize its dynamic expression pattern from gastrulation to early organogenesis. Throughout this developmental period, hsc70 transcripts were largely restricted to neuroectoderm- and mesoderm-derived structures. In stage 10 embryos, Hsc70 protein was expressed in the neural tube with increasing rostrocaudal and decreasing dorsoventral gradients, and in some somite cells. This highly regulated expression of Hsc70 is likely to reflect specific developmental functions, besides its well-characterized role in protein folding.

Apoptotic cell death of proliferating neuroepithelial cells in the embryonic retina is prevented by insulin.

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT-mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor-deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl-3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S-phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl-3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.

Insulin/insulin-like growth factor-I hybrid receptors with high affinity for insulin are developmentally regulated during neurogenesis.

The extensive colocalization of insulin receptor (IR) and insulin-like growth factor-I receptor (IGFR) messenger RNAs during central nervous system development, together with the effects of insulin and IGF-I in neurogenesis, raises the question of how stage- and factor-specific signaling occurs. Thus, it is necessary to characterize the receptor proteins present in vivo to start addressing this issue. Here we have studied the chick embryonic neuroretina at day 6 (E6), when it is predominantly proliferative, and at E12, when neuronal differentiation is advanced. Developmentally regulated high-affinity binding sites for both insulin and IGF-I were detected at E6 and E12. In proliferative neuroretina, typical IGFR with the highest affinity for IGF-I coexisted with separate atypical insulin binding sites, which had similar high affinity for insulin and IGF-I. Immunoprecipitation of ligand-cross-linked receptors with specific antibodies for the IR alpha-subunit, the IR beta-subunit, or the IGFR beta-subunit demonstrated the presence of IR/IGFR hybrids. They were more abundant in E6 than in E12 retina. These hybrid receptors bound most of radiolabeled insulin, but little radiolabeled IGF-I, at tracer concentrations. At E12, the specificity of the insulin binding sites changed, and it was closer to that found with IR in liver, where hybrids were undetectable. The basal autophosphorylation level of these atypical hybrid receptors was high, although insulin and, even more so, IGF-I modestly increased the phosphorylation of two IR beta-subunits of 95 and 105 kDa. The high-affinity/low-discriminative IR/IGFR hybrids predominantly found in a proliferative stage of neurogenesis can mediate the effects of proinsulin and insulin, previously demonstrated in organoculture at this stage. More importantly, this hybrid receptor may be physiologically relevant for the action of the locally produced proinsulin found in early neurogenesis.

Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina.

Regulated preproinsulin gene expression in nonpancreatic tissues during development has been demonstrated in rodents, Xenopus and chicken. Little is known, however, about the synthesis and processing of the primary protein product, proinsulin, in comparison with these events in pancreas. Using specific antisera and immunocytochemistry, immunoblot and HPLC criteria, we characterize the differential processing of proinsulin in developing neuroretina, liver and pancreas. The chick embryo pancreas expresses the convertase PC2, and largely processes proinsulin to insulin. In contrast, little or no mature PC2 is present in embryonic liver and neuroretina and the (pro)insulin immunoactivity identified is predominantly proinsulin.

Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones.

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández-Sánchez et al. (1994) Eur. J. Neurosci., 6,1801-1810] as the noninducible chaperone heat-shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno-crossreactivity and two-dimensional gel-electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central-to-peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat-shock proteins Hsp40, Hsp60 and Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular and developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools and a further insight on neural precursor heterogeneity, and suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis.

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.