Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS
Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS.
SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model
TGF-beta signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3(-/-), Smad3(+/-) and Smad3(+/+) mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3(-/-) mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3(-/-) mice. We also observe marked increase of NF-kappaB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-beta1 in the vessel wall of Smad3(-/-) mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.
Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that beta-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 microm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.
Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. Here, we investigate this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. We find that the impact of altered autophagy depends on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhanced Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy.
Parents Caring for Adult Children With Serious Mental Illness: A Qualitative Descriptive Study: A Dissertation
The purpose of this study was to examine parents’ management styles when caring for adult children with serious mental illness (SMI), as well as parents’ perspectives on what type of community-based mental health interventions would support and/or enhance overall family functioning. This qualitative descriptive study was undergirded by Knafl and Deatrick’s Family Management Style Framework. Thirty parents (N = 30) caring for adult children with SMI over age 18 were recruited as participants. Demographic data included age, gender, ethnicity, educational level, annual income, and National Alliance on Mental Illness membership. Parents were interviewed in their homes or other private setting. Verbal informed consent was obtained. Audio-recorded, individual, semistructured interviews were conducted until redundancy was achieved. Data were analyzed using qualitative content analysis. Four major themes emerged from the data. These themes described prolonged, difficult, and confusing phases that parents and the family undergo in caring for an adult child with SMI. These phases have a progressive nature, moving from parents recognizing that their child has a SMI to redefining family life as a result of caring for an adult child with SMI. Successful management of these phases must include increasing access to mental health information, mental health screening, early interventions, violence prevention, and various treatment options for adult children and their families.
Breast cancer incidence and mortality are rapidly increasing in low- and middle-income countries like Uganda. Shifting the proportion of women presenting with late-stage breast cancer to early-stage breast cancer (downstaging) at the time of diagnosis would substantially improve survival and efficient use of available resources. Imaging The World (ITW) conducted a pilot study in Uganda where trained village health teams (VHTs) promoted breast cancer awareness in the Kamuli District (Uganda). As a result, 212 women with self-detected lumps presented to the community health center level III (Nawanyago HCIII) for a clinical breast examination (CBE). Patients with masses on CBE were examined with breast ultrasound by a certified sonographer trained in breast imaging. Women with ultrasound-detected masses were referred to a regional health center for further evaluation. Of the 212 women, 44 (21%) had a palpable mass by CBE, 11 (28%) examined by ultrasound were recommended for biopsy, and four breast cancers were diagnosed. Providing ultrasound scanning at Nawanyago HCIII reduced the number of women travelling to the referral hospital by 75%. As a result of breast cancer awareness and ultrasound studies, we were able to diagnose breast cancer at an earlier stage than would be otherwise possible. This pilot project supports locally available breast ultrasound as a resource-appropriate strategy to downstage breast cancer in a low-income country.
This is the April 2016 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
This is the March 2016 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
This is the February 2016 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
This is the January 2016 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
Protection against Experimental Cryptococcosis following Vaccination with Glucan Particles Containing Cryptococcus Alkaline Extracts
A vaccine capable of protecting at-risk persons against infections due to Cryptococcus neoformans and Cryptococcus gattii could reduce the substantial global burden of human cryptococcosis. Vaccine development has been hampered though, by lack of knowledge as to which antigens are immunoprotective and the need for an effective vaccine delivery system. We made alkaline extracts from mutant cryptococcal strains that lacked capsule or chitosan. The extracts were then packaged into glucan particles (GPs), which are purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Subcutaneous vaccination with the GP-based vaccines provided significant protection against subsequent pulmonary infection with highly virulent strains of C. neoformans and C. gattii. The alkaline extract derived from the acapsular strain was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the most abundant proteins were identified. Separation of the alkaline extract by size exclusion chromatography revealed fractions that conferred protection when loaded in GP-based vaccines. Robust Th1- and Th17-biased CD4(+) T cell recall responses were observed in the lungs of vaccinated and infected mice. Thus, our preclinical studies have indicated promising cryptococcal vaccine candidates in alkaline extracts delivered in GPs. Ongoing studies are directed at identifying the individual components of the extracts that confer protection and thus would be promising candidates for a human vaccine.
IMPORTANCE: The encapsulated yeast Cryptococcus neoformans and its closely related sister species, Cryptococcus gattii, are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted from Cryptococcus by treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains of C. neoformans and C. gattii. The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens.
Until recently, a diagnosis of peritoneal carcinomatosis was uniformly accompanied by a grim prognosis that was typically measured in weeks to months. Consequently, the management of carcinomatosis revolves largely around palliation of symptoms such as bowel obstruction, nausea, pain, fatigue, and cachexia. A prior lack of effective treatment options created the nihilistic view that currently exists and persists despite improvements in the efficacy of systemic therapy and the evolution of multimodality approaches including surgery and intraperitoneal chemotherapy. This article reviews the evolution and current state of treatment options for patients with peritoneal carcinomatosis. In addition, it highlights recent advances in understanding the molecular biology of carcinomatosis and the focus of current and future clinical trials. Finally, this article provides practical management options for the palliation of common complications of carcinomatosis. It is hoped that the reader will recognize that carcinomatosis is no longer an imminent death sentence and that through continued research and therapeutic innovation, clinicians can make an even greater impact on this form of metastatic cancer.
A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.
NeuN(+) neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure
Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved. We exposed 26 young adult macaque monkeys for 6months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts. After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups. Chronic clozapine exposure increases the proportion of NeuN(+) nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.