Altered brain and physiological stress responses in early psychosis.

Stress is proposed to be a crucial factor in the onset and presentation of psychosis. The early stage of psychosis provides a window into how stress interacts with the emergence of psychosis. Yet, how people with early psychosis respond to stress remains unclear. The current study examined how stress responses (brain, physiological, self-report) differ in early psychosis. Forty participants (20 early psychosis [EP], 20 healthy controls [HC]) completed a stress task in the scanner that involved viewing stressful and neutral-relaxing images. Physiological responses (cortisol, heart rate) and self-report of stress were also assessed. Region of Interest analyses were conducted with brain regions previously shown to be activated during the stress task (amygdala, hippocampus, striatum, hypothalamus, prefrontal cortex [dorsolateral, ventrolateral, medial orbital]). Linear mixed models were used to test for effects of group (EP, HC) and emotion (stress, neutral-relaxing). HC had higher hippocampus activation to stress versus neutral-relaxing conditions while EP did not show a difference (group x emotion interaction, p = 0.04). There were also significant main effects of group with EP having higher amygdala activation (p = 0.01), ventrolateral prefrontal cortex activation (vlPFC, p = 0.03), self-report of stress (p = 0.01), and heart rate (p < 0.001). Our study found preliminary evidence that people with early psychosis showed heightened response to stressful and non-threatening situations, across multiple levels of stress responses. Our findings suggest a novel perspective on stress alterations in early psychosis and highlight the importance of considering both stressful and non-stressful situations.

Attention-deficit/hyperactivity disorder in youth with psychosis spectrum symptoms.

BACKGROUND: Childhood attention deficit-hyperactivity disorder (ADHD) is common in psychotic disorders. However, prevalence estimates vary widely and the impact of ADHD on the severity of psychotic symptoms and associated features is unclear. We used the Philadelphia Neurodevelopmental Cohort (PNC; n = 9498 youth age 8-21), which includes a comprehensive structured interview of clinical symptoms and the Penn Computerized Neurocognitive Battery (CNB), to clarify the prevalence of ADHD in psychosis spectrum (PS) youth and determine if comorbid ADHD is associated with severity of psychotic symptoms and cognitive impairment. METHODS: Prevalence of ADHD among PS youth was established by comparing PS youth to all other youth in the PNC cohort. Cognition was compared between four groups: typically developing (TD), ADHD, PS without ADHD (PS-ADHD), and PS with ADHD (PS+ADHD). To evaluate the impact of ADHD on psychosis symptomatology, severity of positive and negative psychotic symptoms was compared between PS-ADHD and PS+ADHD groups. RESULTS: ADHD was more prevalent in PS youth compared to non-PS youth (45% vs. 20%). Cognition was significantly impaired in PS youth compared to TD youth, but the presence of ADHD in PS youth was not associated with greater cognitive impairment. Co-morbid ADHD was, however, associated with more severe psychosis symptoms in PS youth. CONCLUSION: ADHD is more common among PS youth compared to youth without PS symptoms and is associated with more severe psychotic symptoms, but not severity of cognitive impairment. The association between ADHD and psychotic disorders may be mediated by psychosis symptoms in youth and may manifest a more stable cognitive impairment.

Novel characteristics identified in two cases of feline cowpox virus infection.

CASE SERIES SUMMARY: This case series discusses novel characteristics identified in two cases of cowpox. One presented with upper airway signs, and was identified to have a focal laryngeal lesion. The other had central neurological signs at the terminal stages, with intracytoplasmic inclusion bodies identified within the cerebral hemispheres on histopathology. RELEVANCE AND NOVEL INFORMATION: Currently, cowpox would be an unlikely consideration in patients with neurological signs or upper respiratory noise. These cases both document novel presentations of cowpox infection, which clinicians should be aware of and consider as differential diagnoses in patients with these atypical presentations.

Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications.

Design and validation of a simulator for equine joint injections.

Joint injections are commonly used in equine practice for diagnosis and treatment of joint disorders. Performing joint injections is hence an essential skill for equine practitioners. However, opportunities for veterinary students to practice this skill are often scarce in veterinary curricula. The aim of this study was to design and validate an equine joint injection simulator. We hypothesized that the simulator will enhance student ability and confidence in performing joint injections. The simulator was constructed around an equine forelimb skeleton with soft tissues rebuilt using building foam and rubber bands. An electrical circuit including a buzzer, a battery, wire wool in the joints, and a hypodermic needle at the end of the cable was incorporated. If the students placed the needle into the joint correctly, instant auditory feedback was provided by the buzzer. To validate the simulator, 45 veterinary students were allocated to three groups: cadaver limb, textbook, or simulator. Students' ability to perform joint injections was tested and students' opinions were evaluated with a questionnaire. The proportion of students performing a metacarpophalangeal (MCP) joint injection correctly was significantly higher in the cadaver (93%) and simulator (76%) groups compared to the textbook group (50%). There was no significant difference between groups for performing a distal interphalangeal (DIP) joint injection correctly. Students rated the learning experience with the cadaver and simulator group high and with the textbook group low. The joint injection simulator represents an affordable teaching aid that allows students to repeatedly practice this skill in their own time with immediate feedback.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Culture of human pluripotent stem cells on glass slides for high-resolution imaging.

For certain applications, particularly experiments involving high-resolution imaging, it is necessary to culture cells on glass slides or cover glasses. This chapter describes techniques for successfully growing human embryonic stem cells (hESCs) on glass surfaces under three different conditions - serum-containing, serum-free, and following single-cell dissociation. It is anticipated that these techniques will extrapolate to other types of pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic germ cells (EGCs).

Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases.

Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells.

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

Cell-cell signaling through NOTCH regulates human embryonic stem cell proliferation.

Unlike pluripotent mouse embryonic stem (ES) cells, human ES cells and their malignant equivalents, embryonal carcinoma (EC) cells, require close cell-cell contact for efficient growth. Signaling through the NOTCH receptor, initiated by interaction with ligands of the DELTA/JAGGED family expressed on neighboring cells, plays a role in regulating the self-renewal of several stem cell systems. Members of the NOTCH and DELTA/JAGGED families are expressed by human EC and ES cells, and we have therefore investigated the possible role of NOTCH in the maintenance of these cells. Cleavage of both NOTCH1 and NOTCH2 to yield the intracellular domain responsible for the canonical signaling pathway of NOTCH was detected in several human EC and ES cell lines, suggesting that NOTCH signaling is active. Furthermore, the proliferation of human EC cells, as well as the expression of several downstream NOTCH target genes, was markedly reduced after small interfering RNA knockdown of NOTCH1, NOTCH2, and the canonical effector CBF-1 or after blocking NOTCH signaling with the gamma-secretase inhibitor L-685,458. The inhibitor also caused a reduction in the growth of human ES cells, although without evidence of differentiation. The results indicate that cell-cell signaling through the NOTCH system provides a critical cue for the proliferation of human EC and ES cell in vitro.

Randomized pilot study of a new atrial-based minimal ventricular pacing mode in dual-chamber implantable cardioverter-defibrillators.

OBJECTIVE: We hypothesized that a new minimal ventricular pacing mode (MVP) that provides AAI/R pacing with ventricular monitoring and back-up DDD/R pacing as needed during AV block (AVB) would significantly reduce cumulative percent ventricular pacing compared to DDD/R. BACKGROUND: Conventional DDD/R mode often results in high cumulative percent ventricular pacing that may adversely affect ventricular function and increase risk of heart failure and atrial fibrillation. METHODS: MVP was made operational in 30 patients with DDD/R implantable cardioverter-defibrillators (ICDs) and no history of AVB. Patients were randomized to one week each in DDD/R and MVP. Holter monitor recordings (ECG, intracardiac electrograms, and event markers) and device diagnostics were analyzed for cumulative % atrial paced (Cum%AP), cumulative percent ventricular pacing, and frequency and duration of DDD/R pacing back-up. Diaries were used to report symptoms. RESULTS: Age of the study population was 61 years +/- 12 years and 83% were male. Baseline PR interval was 204 ms +/- 32 ms and programmed AV intervals (DDD/R) were 200 ms +/- 50 ms (paced)/167 ms +/- 54 ms (sensed). Cum%AP was similar between MVP and DDD/R (47.9 +/- 37 vs 46.3 +/- 36). Cumulative percent ventricular pacing was significantly lower in MVP vs DDD/R (3.79 +/- 16.3 vs 80.6 +/- 33.8, P < .0001). Back-up DDD/R pacing during MVP operation due to transient AVB occurred in 10% of patients (9.3 +/- 7.4 [range 1-15] episodes/patient-day, duration 39.7 minutes +/- 156 minutes). Fifteen percent of AV intervals during MVP operation exceeded 300 ms. No significant symptoms were reported during MVP operation. CONCLUSIONS: MVP dramatically reduced cumulative percent ventricular pacing compared to DDD/R while maintaining AV synchrony and providing sensor-modulated atrial pacing support. Intermittent oscillations between MVP and DDD/R during transient AV block appeared safe and well tolerated.

Human embryonic stem cells: multilineage differentiation and mechanisms of self-renewal.

Human ES (hES) cells are pluripotent stem cells isolated from the inner cell mass (ICM) of blastocysts, with the theoretical capacity to differentiate in vitro to produce all somatic and germ cell types. The diverse differentiation repertoire of hES cells makes them ideal candidates for the generation of tissues for transplantation therapies and drug discovery. However, to realize the full potential of hES cells it will be necessary to characterize the mechanisms that control self-renewal and differentiation into specific cell types. We review here the recent developments to differentiate human ES cell into lineages including neural and cardiac. Further, by reference to the self-renewal system established in murine ES we will discuss the possible mechanisms of self-renewal in hES cells.