No abstract

We retrospectively analyzed electronic medical records of patients with Ehlers-Danlos Syndrome hypermobility type (HEDS), including demographic information, workup, rheumatological diagnoses in order to determine its association with rheumatological conditions. HEDS Patients were stratified according to level of workup received (no additional work (physical exam only) = NWU, limited workup = LWU, comprehensive workup = CWU)). HEDS patients were predominantly female (21:4, F:M). The percentage of patients with at least one rheumatological condition was significantly correlated with level of workup (NWU, 9.2%; LWU, 33.3%, CWU, 67.1%; p-value < 0.0001). The HLA-B27 antigen was more prevalent (p-value < 2.2 × 10–8) in the CWU HEDS patients (23.9%) than in the general population of the United States (6.1%). HEDS with CWU were associated with more rheumatological conditions (i.e. psoriasis, ankylosing spondylitis, rheumatoid arthritis, fibromyalgia) than those with NWU or LWU. In conclusion, HEDS is associated with complicated rheumatological conditions, which are uncovered by comprehensive workup. These conditions require different clinical management strategies than HEDS, and left untreated could contribute to the pain or even physical disability (i.e. joint erosions) in HEDS patients. While the mechanisms underlying these associations are unknown, it is important that all HEDS patients receive adequate workup to ensure a complete clinical understanding for the best care strategy possible.

Mammalian cells are amenable to the study of regulatory mechanisms dictating cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is broadly applicable to other mammalian cells. Its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G) under carefully defined experimental conditions to move together into subsequent phases such as the G and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include counterflow centrifugal elutriation, mitotic shake off, chemically induced cell cycle arrest, and newer live cell methods, such as cell permeable dyes. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization and G phase arrest of cultures by serum down-shift. This procedure has been utilized to examine cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Acute inflammation is a key feature of innate immunity that initiates clearance and repair in infected or damaged tissues. Alternatively, chronic inflammation is implicated in numerous disease processes. The contribution of neuroinflammation to the pathogenesis of neurological conditions, including infection, traumatic brain injury, and neurodegenerative diseases, has become increasingly evident. Potential drivers of such neuroinflammation include toll-like receptors (TLRs). TLRs confer a wide array of functions on different cell types in the central nervous system (CNS). Importantly, how TLR activation affects astrocyte functioning is unclear. In the present study, we examined the role of TLR2/4 signaling on various astrocyte functions (i.e., proliferation, pro-inflammatory mediator production, regulatory mechanisms, etc) by stimulating astrocytes with potent exogenous TLR2/4 agonist, bacterial lipopolysaccharide (LPS). Newborn astrocytes were derived from WT, Tnfα −/− , Il1α −/− / Il1β −/− , and Tlr2 −/− /Tlr4 −/− mice as well as Sprague Dawley rats for all in vitro studies. LpS activated mRNA expression of different pro-inflammatory cytokines and chemokines in time-and concentrationdependent manners, and upregulated the proliferation of astrocytes based on increased 3 H-thymidine update. Following LPS-mediated TLR2/4 activation, TNF-α and IL-1β self-regulated and modulated the expression of pro-inflammatory cytokines and chemokines. Polyclonal antibodies against TNF-α suppressed TLR2/4-mediated upregulation of astrocyte proliferation, supporting an autocrine/paracrine role of tnf-α on astrocyte proliferation. Astrocytes perform classical innate immune functions, which contradict the current paradigm that microglia are the main immune effector cells of the CNS. TNF-α plays a pivotal role in the LpS-upregulated astrocyte activation and proliferation, supporting their critical roles in in cnS pathogenesis. Astrocytes are a specialized subtype of glia present throughout the central nervous system (CNS) 1. These star-shaped cells were initially described by German pathologist Rudolf Virchow as the "connective tissue of the brain". As a result of this dismissive analogy, astrocytes were long regarded as functionally insignificant 2. However, recent studies have revealed that astrocytes actually play diverse and critical roles in both CNS homeostasis and pathophysiology, many of which are currently being uncovered. Importantly, under normal circumstances, astrocytes are quiescent; however, in the presence of pathological stimuli, astrocytes re-enter the cell cycle, undergo morphological and biochemical changes, and produce pro-inflammatory cytokines 3,4. The molecular mechanisms underlying these changes remain unknown. One potential mechanism underlying this transition is the activation of toll-like receptors (TLRs). TLRs are a class of membrane-bound receptors called pattern recognition receptors (PRRs) that trigger innate immune responses. Innate immunity is a highly conserved system that is found in all animals an...

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral “sponge” to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.