Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Macromolecular crowding tunes 3D collagen architecture and cell morphogenesis.

Collagen I is the primary extracellular matrix component of most solid tumors and influences metastatic progression. Collagen matrix engineering techniques are useful for understanding how this complex biomaterial regulates cancer cell behavior and for improving in vitro cancer models. Here, we establish an approach to tune collagen fibril architecture using PEG as an inert molecular crowding agent during gelation and cell embedding. We find that crowding produces matrices with tighter fibril networks that are less susceptible to proteinase mediated degradation, but does not significantly alter matrix stiffness. The resulting matrices have the effect of preventing cell spreading, confining cells, and reducing cell contractility. Matrix degradability and fibril length are identified as strong predictors of cell confinement. Further, the degree of confinement predicts whether breast cancer cells will ultimately undergo individual or collective behaviors. Highly confined breast cancer cells undergo morphogenesis to form either invasive networks reminiscent of aggressive tumors or gland and lobule structures reminiscent of normal breast epithelia. This morphological transition is accompanied by expression of cell-cell adhesion genes, including PECAM1 and ICAM1. Our study suggests that cell confinement, mediated by matrix architecture, is a design feature that tunes the transcriptional and morphogenic state of breast cancer cells.

Macro- and microplastics affect cold-water corals growth, feeding and behaviour.

Plastic contamination is now recognized as one of the most serious environmental issues for oceans. Both macro- and microplastic debris are accumulating in surface and deep waters. However, little is known about their impact on deep marine ecosystems and especially on the deep-sea reefs built by emblematic cold-water corals. The aim of this study was to investigate whether plastics affected the growth, feeding and behaviour of the main engineer species, Lophelia pertusa. Our experiments showed that both micro- and macroplastics significantly reduced skeletal growth rates. Macroplastics induced an increased polyp activity but decreased prey capture rates. They acted as physical barriers for food supply, likely affecting energy acquisition and allocation. Inversely, microplastics did not impact polyp behaviour or prey capture rates, but calcification was still reduced compared to control and in situ conditions. The exact causes are still unclear but they might involve possible physical damages or energy storage alteration. Considering the high local accumulation of macroplastics reported and the widespread distribution of microplastics in the world ocean, our results suggest that plastics may constitute a major threat for reef aggradation by inhibiting coral growth, and thus jeopardise the resilience of cold-water coral reefs and their associated biodiversity.

Preventive effects of minocycline in a neurodevelopmental two-hit model with relevance to schizophrenia.

Maternal immune activation can increase the vulnerability of the offspring to develop neuroimmune and behavioral abnormalities in response to stress in puberty. In offspring of immune-challenged mothers, stress-induced inflammatory processes precede the adult onset of multiple behavioral dysfunctions. Here, we explored whether an early anti-inflammatory intervention during peripubertal stress exposure might prevent the subsequent emergence of adult behavioral pathology. We used an environmental two-hit model in mice, in which prenatal maternal administration of the viral mimetic poly(I:C) served as the first hit, and exposure to sub-chronic unpredictable stress during peripubertal maturation as the second hit. Using this model, we examined the effectiveness of the tetracycline antibiotic minocycline (MINO) given during stress exposure to block stress-induced inflammatory responses and to prevent subsequent behavioral abnormalities. We found that combined exposure to prenatal immune activation and peripubertal stress caused significant deficits in prepulse inhibition and increased sensitivity to the psychotomimetic drugs amphetamine and dizocilpine in adulthood. MINO treatment during stress exposure prevented the emergence of these behavioral dysfunctions. In addition, the pharmacological intervention blocked hippocampal and prefrontal microglia activation and interleukin-1ß expression in offspring exposed to prenatal infection and peripubertal stress. Together, these findings demonstrate that presymptomatic MINO treatment can prevent the subsequent emergence of multiple behavioral abnormalities relevant to human neuropsychiatric disorders with onset in early adulthood, including schizophrenia. Our epidemiologically informed two-hit model may thus encourage attempts to explore the use of anti-inflammatory agents in the early course of brain disorders that are characterized by signs of central nervous system inflammation during development.

Extinction of conditioned taste aversion is related to the aversion strength and associated with c-fos expression in the insular cortex.

Taste aversion learning is a type of conditioning where animals learn to associate a novel taste (conditioned stimulus; CS) with a stimulus inducing symptoms of poisoning or illness (unconditioned stimulus; US). As a consequence animals later avoid this taste, a reaction known as conditioned taste aversion (CTA). An established CTA extinguishes over time when the CS is repeatedly presented in the absence of the US. However, inter-individual differences in CTA extinction do exist. Using a model of behavioral conditioning with saccharin as CS and the immunosuppressant cyclosporine A as US, the present study aimed at further elucidating the factors underlying individual differences in extinction learning by investigating whether extinction of an established CTA is related to the strength of the initially acquired CS-US association. In addition, we analyzed the expression of the neuronal activation marker c-fos in brain structures relevant for acquisition and retrieval of the CTA, such as the insular cortex and the amygdala. We here show that animals, displaying a strong CS-US association during acquisition, maintained a strong CTA during unreinforced CS re-exposures, in contrast to animals with moderate CS-US association. Moreover, the latter animals showed increased c-fos mRNA expression in the insular cortex. Our data indicate that CTA extinction apparently depends on the strength of the initially learned CS-US association. In addition, these findings provide further evidence that the memory for the initial excitatory conditioning and its subsequent extinction is probably stored in those structures that participate in the processing of the CS and the US.

Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs.

Preliminary evidence for a modulation of fetal dopaminergic development by maternal immune activation during pregnancy.

Maternal infection during pregnancy is an environmental risk factor for the offspring to develop severe brain disorders, including schizophrenia and autism. However, only little is known about the neurodevelopmental mechanisms underlying the association between prenatal exposure to infection and the emergence of brain and behavioral dysfunctions in later life. Using a mouse model of prenatal immune challenge by the viral mimic polyriboinosinic-polyribocytidilic acid (PolyI:C), we explored the acute effects of maternal immune activation during pregnancy on the development of the fetal dopaminergic system, a neurotransmitter system known to be affected in schizophrenia and related disorders. We found that maternal immunological stimulation in early/middle pregnancy increased the number of mesencephalic dopamine neurons in the fetal brain at middle/late and late gestation. This effect was paralleled by changes in fetal expression of several genes known to be involved in dopamine neuron development, including the inductive signals sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8), as well as transcription factors Nurr1 and Pitx3. These findings provide initial in vivo evidence for a modulation of fetal dopaminergic development by maternal immune activation during pregnancy. Additional investigations of the neurodevelopmental effects of prenatal immune challenge are thus clearly warranted in order to further validate whether abnormal dopaminergic development may be a critical neuropathological mechanism underlying the precipitation of schizophrenia-like brain and behavioral dysfunctions emerging after in utero exposure to infection.

Weakened [corrected] taste-LPS association during endotoxin tolerance.

In naive individuals, the administration of bacterial lipopolysaccharide (LPS) provokes a rapid systemic increase in pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, inducing an acute phase response including sickness behavior. Strong associative learning occurs when relevant gustatory/olfactory stimuli precede the activation of the immune system, affecting long-term individual food selection and nutritional strategies. Repeated LPS administration results in the development of an endotoxin tolerance status, characterized by a drastic reduction in the LPS-induced cytokine response. Here we investigated how the postprandial categorization of a relevant taste (0.2% saccharin) changed after administration of a high dose of LPS (0.5 mg/kg i.p.) in LPS-tolerant animals. Determination of the consummatory fluid intake revealed that, in contrast to LPS-naive rats, taste-LPS association did not occur during endotoxin tolerance. Ninety minutes after the single association trial, the plasma responses of TNF-alpha, IL-1beta and IL-6 were completely blunted in LPS-tolerant animals, which also resulted in low LPS-adipsogenic and LPS-anorexic effects. These findings indicate that an identical immune challenge can result in completely different neuro-behavioral consequences depending on the immune history of the individual, thus revealing part of the complex interconnection between the immune and neuro-endocrine systems in regulating food selection and consumption during the infectious process.

Solution structure of human immunodeficiency virus type 1 Vpr(13-33) peptide in micelles.

Human immunodeficiency virus type 1 protein R (HIV-1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G2 cell cycle arrest and is involved in cellular differentiation and cell death. Also, Vpr subcellular localization is as variable as its functions. It is known that, consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr obviously is dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 13-33 of HIV-1 Vpr in micelles. Employing nuclear magnetic resonance and circular dichroism spectroscopy we found this part of Vpr, known to be essential for nuclear localization, to be almost completely alpha helical. Our results provide structural data suggesting residues 13-33 of Vpr to form an amphipathic, leucine-zipper-like alpha helix that serves as a basis for interactions with a variety of viral and cellular factors.

Working with families of suddenly and critically ill children: physician experiences.

OBJECTIVE: To describe physicians' experiences in attempting to provide optimal care for families of children who suffer from sudden, acute life-threatening conditions (SALTC). DESIGN: To generate descriptive data in this exploratory study, we used qualitative methods including focus groups and in-depth interviews. Transcripts of focus groups and interviews were analyzed for content using standard phenomenologic analysis methods, which resulted in a participant-generated conceptual model of optimal care for families of children with SALTC. SETTING: The intensive care unit of an urban pediatric teaching hospital. PARTICIPANTS: Twenty-two pediatric intensive care unit physicians, including residents, fellows, and attendings. INTERVENTION: None. MAIN OUTCOME MEASURES: Each participating physician provided qualitative descriptions of experiences caring for families of children with SALTC. RESULTS: Physicians identified 4 components of optimal care for families: (1) providing timely, accurate information about their child; (2) maintaining privacy for confidential discussions and personal grieving; (3) giving adequate emotional support; and (4) granting family members the right to hold and comfort their dying child. Physicians also described barriers to, and facilitators of this optimal care. CONCLUSIONS: Descriptive information provided in this exploratory study offers a complex model of optimal family care. Issues that affect the quality of care to families include those related to the context of providing care in a large teaching hospital, as well as subtleties of communication between parents and staff. Physicians' beliefs about optimal care of families in the pediatric intensive care unit revealed implications for both practice and training in pediatrics.

Saying goodbye in the intensive care unit: helping caregivers grieve.

The need for critical care caregivers to complete their own grief work when a patient dies deserves special attention. Positive coping with grief and loss can be encouraged through formal programs and individual and group memorial services.

Verifying endotracheal tube placement with the TRACH ++ MATE intubation system.

Determining the location of the endotracheal tube in critically ill infants and children often creates uncertainty for critical care nurses. A newly developed endotracheal tube system (TRACH MATE) improves confidence in assessing endotracheal tube placement when used appropriately.

Dual-pedicle dermoparenchymal mastopexy.

Mastopexy for treatment of breast ptosis, with or without augmentation or reduction, is often followed by recurrent ptosis. A new mastopexy technique is described which appears to offer long-term correction. After conservative resection of excess skin, the breast parenchyma is elevated from the chest wall, and redundant caudal deepithelialized breast tissue is divided into two equal (or unequal) superiorly based pedicles. These are criss-crossed (as in folding of arms), overlapped, and secured to the pectoral fascia in a position cephalad to the nipple-areolar complex. This technique, dual-pedicle dermoparenchymal mastopexy (DPM), forms a cone of the breast tissue and provides a "cradle" of support. It permits insertion of a prosthesis if needed. Based, in part, on concepts of traditional and more recently described pedicled breast reductions, it enjoys the advantage of preserving skin attachment to underlying unresected breast parenchyma. In addition, it repositions ptotically displaced breast parenchyma into a cephalad position and fixes it (the "pexy") to the chest wall. A 10-year experience is presented with representative cases to illustrate the basic mastopexy and its use with augmentation or reduction.