Gene Expression in Uterine Leiomyoma from Tumors Likely to Be Growing (from Black Women over 35) and Tumors Likely to Be Non-Growing (from White Women over 35)

The study of uterine leiomyomata (fibroids) provides a unique opportunity to investigate the physiological and molecular determinants of hormone dependent tumor growth and spontaneous tumor regression. We conducted a longitudinal clinical study of premenopausal women with leiomyoma that showed significantly different growth rates between white and black women depending on their age. Growth rates for leiomyoma were on average much higher from older black women than for older white women, and we now report gene expression pattern differences in tumors from these two groups of study participants. Total RNA from 52 leiomyoma and 8 myometrial samples were analyzed using Affymetrix Gene Chip expression arrays. Gene expression data was first compared between all leiomyoma and normal myometrium and then between leiomyoma from older black women (age 35 or older) and from older white women. Genes that were found significant in pairwise comparisons were further analyzed for canonical pathways, networks and biological functions using the Ingenuity Pathway Analysis (IPA) software. Whereas our comparison of leiomyoma to myometrium produced a very large list of genes highly similar to numerous previous studies, distinct sets of genes and signaling pathways were identified in comparisons of older black and white women whose tumors were likely to be growing and non-growing, respectively. Key among these were genes associated with regulation of apoptosis. To our knowledge, this is the first study to compare two groups of tumors that are likely to have different growth rates in order to reveal molecular signals likely to be influential in tumor growth.     

Bone morphogenetic protein (BMP) type II receptor is required for BMP-mediated growth arrest and differentiation in pulmonary artery smooth muscle cells

Bone morphogenetic protein (BMP) signals regulate the growth and differentiation of diverse lineages. The association of mutations in the BMP type II receptor (BMPRII) with idiopathic pulmonary arterial hypertension suggests an important role of this receptor in vascular remodeling. Pulmonary artery smooth muscle cells lacking BMPRII can transduce BMP signals using ActRIIa (Activin type II receptor). We investigated whether or not BMP signaling via the two receptors leads to differential effects on vascular smooth muscle cells. BMP4, but not BMP7, inhibited platelet-derived growth factor-activated proliferation in wild-type pulmonary artery smooth muscle cells, whereas neither ligand inhibited the growth of BMPRII-deficient cells. Adenoviral gene transfer of BMPRII enabled BMP4, as well as BMP7, to inhibit proliferation in BMPRII-deficient cells. BMP-mediated growth inhibition was also reconstituted by the BMPRII short isoform, lacking the C-terminal domain present in the long form. BMP4, but not BMP7, induced the expression of osteoblast markers in wild-type cells, whereas neither ligand induced these markers in BMPRII-deficient cells. Overexpression of short or long forms of BMPRII in BMPRII-deficient cells enabled BMP4 and BMP7 to induce osteogenic differentiation. Although signaling via BMPRII or ActRIIa transiently activated SMAD1/5/8, only BMPRII signaling led to persistent SMAD1/5/8 activation and sustained increases in Id1 mRNA and protein expression. Pharmacologic blockade of BMP type I receptor function within 24 h after BMP stimulation abrogated differentiation. These data suggest that sustained BMP pathway activation, such as that mediated by BMPRII, is necessary for growth and differentiation control in vascular smooth muscle.     

Multifunctional human immunodeficiency virus (HIV) gag-specific CD8+ T-cell responses in rectal mucosa and peripheral blood mononuclear cells during chronic HIV type 1 infection

The intestinal tract is a lymphocyte-rich site that undergoes severe depletion of memory CD4(+) T cells within days of simian immunodeficiency virus or human immunodeficiency virus type 1 (HIV-1) infection. An ensuing influx of virus-specific CD8(+) T cells, which persist throughout the chronic phase of infection, has also been documented in the gastrointestinal tract. However, little is known of the functionality of these effector cells or their relationship to the disease course. In this study, we measured CD8(+) T-cell responses to HIV-1 peptides in paired rectal and blood samples from chronically infected patients. In both blood and rectum, there was an immunodominant CD8(+) T-cell response to HIV Gag compared to Pol and Env (P < 0.01). In contrast, cytomegalovirus pp65 peptides elicited gamma interferon (IFN-gamma) secretion strongly in peripheral blood mononuclear cells (PBMC) but weakly in rectal CD8(+) T cells (P = 0.015). Upon stimulation with HIV peptides, CD8(+) T cells from both sites were capable of mounting complex responses including degranulation (CD107 expression) and IFN-gamma and tumor necrosis factor alpha (TNF-alpha) production. In rectal tissue, CD107 release was frequently coupled with production of IFN-gamma or TNF-alpha. In patients not on antiretroviral therapy, the magnitude of Gag-specific responses, as a percentage of CD8(+) T cells, was greater in the rectal mucosa than in PBMC (P = 0.054); however, the breakdown of responding cells into specific functional categories was similar in both sites. These findings demonstrate that rectal CD8(+) T cells are capable of robust and varied HIV-1-specific responses and therefore likely play an active role in eliminating infected cells during chronic infection.     

Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

A Pharmacological Screening Approach for Discovery of Neuroprotective Compounds in Ischemic Stroke

With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-β-hydroxysteroid dehydrogenase-1 (11β-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11β-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.     

Fungal Chitin Dampens Inflammation through IL-10 Induction Mediated by NOD2 and TLR9 Activation

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.

Authors Summary

Chitin is the second most abundant polysaccharide in nature after cellulose and an essential component of the cell wall of all fungal pathogens. The discovery of human chitinases and chitinase-like binding proteins indicates that fungal chitin is recognised by cells of the human immune system, shaping the immune response towards the invading pathogen. We show that three immune cell receptors– the mannose receptor, NOD2 and TLR9 recognise chitin and act together to mediate an anti-inflammatory response via secretion of the cytokine IL-10. This mechanism may prevent inflammation-based damage during fungal infection and restore immune balance after an infection has been cleared. By increasing the chitin content in the cell wall pathogenic fungi may influence the immune system in their favour, by down-regulating protective inflammatory immune responses. Furthermore, gene mutations and dysregulated enzyme activity in the described chitin recognition pathway are implicated in inflammatory conditions such as Crohn''s Disease and asthma, highlighting the importance of the discovered mechanism in human health.     

Myocardial dysfunction in an animal model of cancer cachexia

AimsFatigue is a common occurrence in cancer patients regardless of tumor type or anti-tumor therapies and is an especially problematic symptom in persons with incurable tumor disease. In rodents, tumor-induced fatigue is associated with a progressive loss of skeletal muscle mass and increased expression of biomarkers of muscle protein degradation. The purpose of the present study was to determine if muscle wasting and expression of biomarkers of muscle protein degradation occur in the hearts of tumor-bearing mice, and if these effects of tumor growth are associated with changes in cardiac function.Main methodsThe colon26 adenocarcinoma cell line was implanted into female CD2F1 mice and skeletal muscle wasting, in vivo heart function, in vitro cardiomyocyte function, and biomarkers of muscle protein degradation were determined.Key findingsExpression of biomarkers of protein degradation were increased in both the gastrocnemius and heart muscle of tumor-bearing mice and caused systolic dysfunction in vivo. Cardiomyocyte function was significantly depressed during both cellular contraction and relaxation.SignificanceThese results suggest that heart muscle is directly affected by tumor growth, with myocardial function more severely compromised at the cellular level than what is observed using echocardiography.     

Territorial behaviour of the red-backed salamander: Expulsion of intruders

Red-backed salamanders, Plethodon cinereus, were paired in laboratory chambers where they defended areas against each other by threat display and biting. Difference in sizes of paired individuals was not correlated with which one would enter the other's area or be more aggressive after an intrusion. We also found no sexual differences in agonistic behaviour. Both defenders and intruders assumed a threat posture for about 50% of the invasion periods, but defenders exhibited a higher rate of biting. Defenders expelled intruders in 74% of all encounters, while intruders expelled defenders in 18%, with 8% of the encounters a draw. Intruders frequently left the contested area even in the absence of biting or threat display by the defender, apparently in response to the defender's pheromones or to the high potential cost of an escalated aggressive contest (damage to the chemosensory structures). These data provide behavioural evidence that a salamander can evict intruders from a defended area and thus support the hypothesis of territoriality among terrestrial salamanders.