Poor body condition is associated with lower hippocampal plasticity and higher gut methanogen abundance in adult laying hens from two housing systems.

It is still unclear which commercial housing system provides the best quality of life for laying hens. In addition, there are large individual differences in stress levels within a system. Hippocampal neurogenesis or plasticity may provide an integrated biomarker of the stressors experienced by an individual. We selected 12 adult hens each with good and poor body condition (based on body size, degree of feather cover and redness of the comb) from a multi-tier free range system containing H&N strain hens, and from an enriched cage system containing Hy-Line hens (n = 48 total). Immature neurons expressing doublecortin (DCX) were quantified in the hippocampus, contents of the caecal microbiome were sequenced, and expression of inflammatory cytokines was measured in the spleen. DCX+ cell densities did not differ between the housing systems. In both systems, poor condition hens had lower DCX+ cell densities, exhibited elevated splenic expression of interleukin-6 (IL6) mRNA, and had a higher relative caecal abundance of methanogenic archea Methanomethylophilaceae. The findings suggest poor body condition is an indicator that individual hens have experienced a comparatively greater degree of cumulative chronic stress, and that a survey of the proportion of hens with poor body conditions might be one way to evaluate the impact of housing systems on hen welfare.

Keel bone fractures induce a depressive-like state in laying hens.

In commercial flocks of laying hens, keel bone fractures (KBFs) are prevalent and associated with behavioural indicators of pain. However, whether their impact is severe enough to induce a depressive-like state of chronic stress is unknown. As chronic stress downregulates adult hippocampal neurogenesis (AHN) in mammals and birds, we employ this measure as a neural biomarker of subjective welfare state. Radiographs obtained longitudinally from Lohmann Brown laying hens housed in a commercial multi-tier aviary were used to score the severity of naturally-occurring KBFs between the ages of 21-62 weeks. Individual birds' transitions between aviary zones were also recorded. Focal hens with severe KBFs at 3-4 weeks prior to sampling (n = 15) had lower densities of immature doublecortin-positive (DCX+) multipolar and bipolar neurons in the hippocampal formation than focal hens with minimal fractures (n = 9). KBF severity scores at this time also negatively predicted DCX+ cell numbers on an individual level, while hens that acquired fractures earlier in their lives had fewer DCX+ neurons in the caudal hippocampal formation. Activity levels 3-4 weeks prior to sampling were not associated with AHN. KBFs thus lead to a negative affective state lasting at least 3-4 weeks, and management steps to reduce their occurrence are likely to have significant welfare benefits.

Unpredictable Chronic Mild Stress Suppresses the Incorporation of New Neurons at the Caudal Pole of the Chicken Hippocampal Formation.

In the mammalian brain, adult hippocampal neurogenesis (AHN) is suppressed by chronic stress, primarily at the ventral pole of the hippocampus. Based upon anatomy, we hypothesise that the caudal pole of the avian Hippocampal Formation (HF) presents a homologous subregion. We thus investigated whether AHN is preferentially suppressed in the caudal chicken HF by unpredictable chronic mild stress (UCMS). Adult hens were kept in control conditions or exposed to UCMS for 8 weeks. Hens experiencing UCMS had significantly fewer doublecortin-positive multipolar neurons (p < 0.001) and beaded axons (p = 0.021) at the caudal pole of the HF than controls. UCMS birds also had smaller spleens and lower baseline plasma corticosterone levels compared to controls. There were no differences in AHN at the rostral pole, nor were there differences in expression of genetic mediators of the HPA stress response in the pituitary or adrenal glands. Duration of tonic immobility and heterophil/lymphocyte (H/L) ratios were also not responsive to our UCMS treatment. These results support the hypothesised homology of the caudal pole of the avian HF to the ventral pole of the rodent hippocampus. Furthermore, quantifying neurogenesis in the caudal HF post-mortem may provide an objective, integrative measure of welfare in poultry, which may be more sensitive than current welfare measures.

Broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency.

Chronic placental insufficiency and subsequent intrauterine growth restriction (IUGR) increase the risk of hypoxic-ischemic encephalopathy in the newborn by 40 fold. The latter, in turn, increases the risk of cerebral palsy and developmental disabilities. This study seeks to determine the effectiveness of broccoli sprouts (BrSp), a rich source of the isothiocyanate sulforaphane, as a neuroprotectant in a rat model of chronic placental insufficiency and IUGR. Placental insufficiency and IUGR was induced by bilateral uterine artery ligation (BUAL) on day E20 of gestation. Dams were fed standard chow or chow supplemented with 200mg of dried BrSp from E15 - postnatal day 14 (PD14). Controls received Sham surgery and the same dietary regime. Pups underwent neurologic reflex testing and open field testing, following which they were euthanized and their brains frozen for neuropathologic assessment. Compared to Sham, IUGR pups were delayed in attaining early reflexes and performed worse in the open field, both of which were significantly improved by maternal supplementation of BrSp (p<0.05). Neuropathology revealed diminished white matter, ventricular dilation, astrogliosis and reduction in hippocampal neurons in IUGR animals compared to Sham, whereas broccoli sprout supplementation improved outcome in all histological assessments (p<0.05). Maternal dietary supplementation with BrSp prevented the detrimental neurocognitive and neuropathologic effects of chronic intrauterine ischemia. These findings suggest a novel approach for prevention of cerebral palsy and/or developmental disabilities associated with placental insufficiency.

Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the ß-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Helpful communications during the diagnostic period: an interpretive description of patient preferences.

With a diagnosis of cancer, life changes for patients in a profound manner. The window of time known as cancer diagnosis is one of considerable turbulence and distress for patients. Therefore, diagnosis constitutes a time during which communication with healthcare professionals is of particular importance in setting the stage for the way cancer illness will be experienced. Our research explores communications throughout the cancer trajectory from the perspective of patients themselves. We are following a sample of 60 cancer patients, representing a range of tumour sites, from the early diagnostic period through to recovery, chronic, or advanced disease. Using interpretive description analysis techniques, we document patterns and themes related to various components of the cancer journey. In this paper, we focus on themes related to perceived helpful communication during the diagnosis experience as reported by our study participants both at the time of being newly diagnosed patients, and as they reflect on that period 12 months later. These findings illuminate experiential issues of importance to patients in relation to cancer care communication and the manner in which helpful communications during this sensitive time may facilitate the subsequent experience living with and obtaining care for cancer.

Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members.

The epidermal growth factor receptor (EGFR) is a central regulator of proliferation and progression in human cancers. Five EGFR inhibitors, two monoclonal antibodies and three TKIs, have recently gained FDA approval in oncology (cetuximab, panitumumab, erlotinib, gefitinib and lapatinib). These strategies of EGFR inhibition demonstrate major tumor regressions in approximately 10-20% of advanced cancer patients. However, many tumors eventually manifest acquired resistance to treatment. In this study we established and characterized a model to study molecular mechanisms of acquired resistance to the EGFR monoclonal antibody cetuximab. Using high-throughput screening we examined the activity of 42 receptor tyrosine kinases in resistant tumor cells following chronic exposure to cetuximab. Cells developing acquired resistance to cetuximab exhibited increased steady-state EGFR expression secondary to alterations in trafficking and degradation. In addition, cetuximab-resistant cells manifested strong activation of HER2, HER3 and cMET. EGFR upregulation promoted increased dimerization with HER2 and HER3 leading to their transactivation. Blockade of EGFR and HER2 led to loss of HER3 and PI(3)K/Akt activity. These data suggest that acquired resistance to cetuximab is accompanied by dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3. Taken together these findings suggest a rationale for the clinical evaluation of combinatorial anti-HER targeting approaches in tumors manifesting acquired resistance to cetuximab.

The unexpected preference for the fac-isomer with the tris(5-ester-substituted-2,2'-bipyridine) complexes of ruthenium(II).

The synthesis of a number of new 2,2'-bipyridine ligands, functionalized with bulky ester side groups, is reported (L2-L8). Their reaction with [Ru(DMSO)(4)Cl(2)] gives rise to tris-chelate ruthenium(II) metal complexes which show an unusually high proportion of the fac-isomer, as judged by (1)H NMR following conversion to the ruthenium(II) complex of 2,2'-bipyridine-5-carboxylic acid methyl ester (L1). The initial reaction appears to have thermodynamic control with the steric bulk of the ligands causing the third ligand to be labile under the reaction conditions used, giving rise to disappointing yields and allowing rearrangement to the more stable facial form. DFT studies indicate that this does not appear to be as a consequence of a metal centered electronic effect. The two isomers of [Ru(L1)(3)](PF(6))(2) were separated into the two individual forms using silica preparative plate chromatographic procedures, and the photophysical characteristics of the two forms compared. The results appear to indicate that there is no significant difference in both their room temperature electronic absorption and emission spectra or their excited state lifetimes at 77 K.

Prolonged seizures exacerbate perinatal hypoxic-ischemic brain damage.

This study was undertaken to clarify whether seizures in the newborn cause damage to the healthy brain and, more specifically, to determine the extent to which seizures may contribute to the brain-damaging effects of hypoxia-ischemia (HI). Seizures were induced in 10-d-old rat pups with kainic acid (KA). Seizure duration was determined electrographically. HI was induced by common carotid artery ligation followed by exposure to 8% oxygen for either 15 or 30 min. Six groups of animals were assessed: 1) controls [neither KA nor HI (group I)]; 2) group II, KA alone; 3) group III, 15 min HI alone; 4) group IV,15 min HI plus KA; 5) group V, 30 min HI alone; and 6) group VI, 30 min HI plus KA. Animals were assessed neuropathologically at 3 (early) and 20 (late) d of recovery. KA injection without hypoxia resulted in continuous clinical and electrographic seizures lasting a mean of 282 min. No neuropathologic injury was seen in groups I (no HI or KA), II (KA alone), III (15 min HI alone), or IV (15 min HI and KA). Animals in group V (30 min HI alone) displayed brain damage with a mean score of 2.3 and 0.60 at 3 and 20 d of recovery, respectively. Animals in group VI (30 min HI and KA) had a mean score of 12.1 and 3.65 at 3 and 20 d of recovery, respectively. Compared with group V, the increased damage as a result of the seizure activity in group VI occurred exclusively in the hippocampus. Status epilepticus in the otherwise "healthy" neonatal brain does not cause neuropathologic injury. However, seizures superimposed on HI significantly exacerbate brain injury in a topographically specific manner.

A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf.

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.

Long-term gastrostomy in children: caregiver coping.

As technologic interventions such as long-term gastrostomy in children with severe disability become commonplace, and increasing numbers of medically complex children are cared for in the home, nurses will require specialized knowledge to support families. In this article, the authors document findings from a longitudinal study of families caring for a child with a gastrostomy, specifically addressing the caregiver perspective on how families cope with this challenge. The findings reveal a complex set of effective coping strategies as articulated by family caregivers experienced in the management of long-term gastrostomy. Further, these caregivers reveal insights about gastrostomy's technical, social, and psychologic implications for both themselves and their children. In documenting an insider perspective on this phenomenon, this study provides nurses with a comprehensive orientation to the ways in which healthcare professionals can better support family caregiver coping.

Differential binding and internalization of insulin-like growth factor (IGF)-I in cultured human trophoblast and JEG-3 cells: possible modulatory effect of IGF binding proteins (BP).

The present study was undertaken to characterize and identify the insulin-like growth factor binding proteins (IGF BPs) secreted by placental cells and their possible modulatory effect on IGF-I binding to cell surface receptors. The experimental approach taken was comparative characterization of binding and internalization of IGF-I and its analog, [Gln(3), Ala(4), Tyr(15), Leu(16) (QAYL)]IGF-I, with reduced affinity for IGF BP, in two different placental cell culture models. One was human placental trophoblast in primary culture and the other, JEG-3 cells, a human choriocarcinoma cell line, representing placental trophoblasts. Binding of [(125)I]IGF-I in both trophoblast and JEG-3 cells was time and temperature dependent. At 37°C, the plateau of [(125)I]IGF-I binding to both the cells (1-2% specific binding per 10(5) cells) was reached by 40-60 min. At 4°C, the time required to reach the plateau in both cells was increased to ∼4h. The maximum binding of [(125)I]IGF-I to trophoblasts, however, was ∼2 times higher than at 37°C, whereas in JEG-3 cells binding remained the same. Internalization of [(125)I]IGF-I in trophoblast cells was low and temperature independent. At both 37 and 4°C, ≤30% of the total cell-associated [(125)I]IGF-I was internalized. In contrast, internalization of [(125)I]IGF-I in JEG-3 cells was rapid and temperature dependent. At 37°C, ≥60% of the total cell-associated [(125)]IGF-I was internalized by 40-60min. At 4°C, internalization was slow and did not exceed 10% of the total cell-associated radioactivity. Binding of [(125)I-QAYL]IGF-I to trophoblasts, in comparison to [(125)I]IGF-I, was significantly different. The binding was undetectable at 37°C and it was low at 4°C. In JEG-3 cells, however, the binding and internalization of [(125)I]-QAYL]IGF-I at both the temperatures were comparable to that of [(125)I]IGF-I. Further characterization of the two [(125)I]IGF-I bindings to the different placental cells was achieved by binding competition studies using unlabelled IGF-I, [QAYL]IGF-I and [Leu]IGF-I, another analog of IGF-I, [Leu(24), 1-62]IGF-I with reduced affinity for the IGF-I receptor, and α-IR3, a monoclonal antibody to the IGF-I receptor. The different potencies of IGF-I and its analogs, and α-IR3 in competing binding of two [(125)I]IGF-Is in the different cells suggested that binding of IGF-I to JEG-3 cells was predominantly to IGF-I receptor, whereas to trophoblast cells it was to IGF BP. This was confirmed by affinity cross-linking studies. The major affinity cross-linked [(125)I]IGF-I complex in trophoblast cells was shown to be a protein of Mr. ∼43 kDa, corresponding to IGF BP-3. In JEG-3 cells, the major cross-linked [(125)I]IGF-I and-[QAYL]IGF-I complexes were proteins of Mr ∼130 kDa and >260 kDa, corresponding to the monomeric and multimeric forms of IGF-I receptor. The ∼43 kDa complex in trophoblast was confirmed to be IGF BP-3 by identification of the characteristics of the IGF BP secreted by trophoblast by Western ligand and immunoblots of the conditioned media. JEG-3 cells did not secrete IGF BP. In conclusion, the membrane associated IGF BP-3 in trophoblast cells, shown here, imply anin vivo modulatory effect of membrane bound IGF BP-3 on IGF-I action in placenta. JEG-3 cells, not secreting IGF-BP, offer an attractive model to study the interactive mechanism of IGF-I and IGF BP-3 actions on the placenta.

Role of cations and IgA in saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis patients.

Oral colonization by Pseudomonas aeruginosa possibly precedes the pulmonary infection process in cystic fibrosis (CF) patients. As bacterial aggregates may play a role in establishment of pulmonary infections, involvement of IgA and cations in CF patient saliva-mediated aggregation of P. aeruginosa was investigated. For colonized patients, P. aeruginosa aggregation correlated with bacterial-specific and total salivary IgA. Cation or IgA depletion reduced P. aeruginosa aggregation by saliva from all patients. However, if cations were removed before IgA, and saliva was then reconstituted with calcium, only colonized patient saliva showed reduced aggregation. Aggregation by IgA-depleted saliva was augmented by reconstituting with original IgA. CF patient saliva-mediated aggregation of P. aeruginosa thus is cation-dependent and enhanced by bacterial-specific IgA. Characterizing the interactions among bacterial aggregating factor(s), cations, and antibodies in CF saliva will help clarify the link between P. aeruginosa oral colonization and pulmonary infections in CF patients.

Renal cystic disease in childhood.

The authors present a comprehensive review of the diagnostic features of eight forms of renal cystic disease that occur in childhood. Sonographic findings are emphasized.

Benign suprasellar cysts: the CT approach.

Preoperative diagnosis of intracranial cysts has been simplified and made more rapid and accurate with computed tomography (CT). By means of conventional CT and CT metrizamide ventriculography, the position and communication of intracranial cysts with the ventricular system and subarachnoid space or cisterns can be demonstrated. Suprasellar arachnoid cysts can produce significant neurologic and endocrinologic abnormalities due to their position. They are a surgically curable cause of hydrocephalus. Preoperative differentiation from aqueduct stenosis or other causes of a large third ventricle is important. The usefulness of coronal CT and CT metrizamide ventriculography in the investigation of these lesions is illustrated in six patients.

CT of neuroblastomas and ganglioneuromas in children.

Computed tomography (CT) has made a profound impact on the diagnosis and treatment of neuroblastomas and ganglioneuromas. The size, location, calcification, composition, and contiguous spread of the tumors has been well demonstrated by CT. CT is essential for their staging, subsequent treatment, and follow-up. Seventy-seven children were reviewed, 67 with neuroblastoma and 10 with ganglioneuroma seen between 1976 and 1980. Fifty-eight had one or more body CT scans, 22 had metrizamide myelography and/or CT metrizamide myelography, and three patients had cranial CT. Intraspinal extension of tumor occurred in 11 instances, several requiring decompressive surgery. A workup plan for optimal use of CT and CT metrizamide myelography was developed from this experience.