Citations

Abstract β -glucans represent major structural components of fungal cell walls. We recently reported that Pneumocystis carinii β -glucans stimulate alveolar macrophages to release proinflammatory cytokines. Macrophage activation by β -glucan is augmented by serum, implying the presence of circulating factors that interact with β -glucans and enhance their ability to stimulate macrophages. Using β -glucan–enriched cell wall fractions from P. carinii and Saccharomyces cerevisiae, two prominent proteins were precipitated from serum and demonstrated to be vitronectin (VN) and fibronectin (FN) by immune analysis. Preincubation of β -glucan with VN or FN enhanced macrophage activation in response to this cell wall component. Because VN and FN accumulate in the lungs during P. carinii pneumonia, we further investigated hepatic and pulmonary expression of VN and FN messenger RNA during infection. P. carinii pneumonia in rodents is associated with increased hepatic expression of VN and FN as well as increased local expression of FN in the lung. Because interleukin (IL)-6 represents the major regulator of VN and FN expression during inflammatory conditions, we measured macrophage IL-6 release in response to stimulation with P. carinii β -glucan. Stimulation of macrophages with P. carinii β -glucan induced significant release of IL-6. Elevated concentrations of IL-6 were noted in the blood of infected animals compared with uninfected control animals. These studies indicate that VN and FN bind to β -glucan components of P. carinii and augment macrophage inflammatory responses. P. carinii cell wall β -glucan stimulates secretion of IL-6 by macrophages, thereby enhancing hepatic synthesis of both VN and FN, and lung synthesis of FN during pneumonia.

Abstract Pneumocystis carinii continues to cause severe pneumonia in immunocompromised patients. Surfactant protein D (SP-D), a lung collectin, markedly accumulates during P. carinii pneumonia and binds to glycoprotein A (gpA) on the surface of P. carinii, thereby enhancing interactions with alveolar macrophages. Herein, we report the structural basis of the interaction of SP-D with gpA. We demonstrate that natural SP-D binds to purified gpA in the presence of 2 mM calcium in a saturable, concentration-dependent manner, which is abolished by 10 mM ethylenediaminetetraacetic acid. Increasing concentrations of calcium under otherwise cation-free conditions significantly enhance SP-D binding to gpA, whereas manganese and magnesium cations have minimal effect. Maximal SP-D binding occurs at pH 7.4, with significant inhibition at pH 4. SP-D binding to gpA is also competitively inhibited by maltose > glucose > mannose > N-acetyl-glucosamine. Comparison of the binding of various natural and recombinant forms of SP-D to gpA reveals that the number of carbohydrate recognition domains (CRDs) in a given SP-D form determines the relative extent of binding to gpA. Maximal binding is observed with natural SP-D (dodecamers and higher order SP-D complexes) followed by recombinant dodecamers. In contrast, recombinant full-length trimers exhibit substantially less binding, which is similar to that observed with a recombinant truncated molecule consisting of the CRD and neck regions, and containing trimers of this portion of the molecule. Taken together, these findings strongly indicate that the CRD of SP-D mediates interaction with P. carinii gpA through its attached oligosaccharides and that the extent of SP-D binding to P. carinii is greatest with dodecamers and higher order forms of SP-D.

Abstract The eosinophil-associated ribonuclease (Ear) family in the mouse consists of thirteen genes, eleven of which encode RNases that have physical/functional properties similar to the human Ears, eosinophil-derived neurotoxin and eosinophil cationic protein. The expression of Ear genes in the mouse is confined to sites of known eosinophilopoiesis, with the exception of the lung. Two Ear genes, Ear1 and Ear2, are predominantly expressed in the lungs of naive mice. Total Ear gene expression and RNase activity in bronchoalveolar lavage fluid increases significantly upon the induction of pulmonary inflammation using an ovalbumin (OVA) model of allergic sensitization and challenge. Interestingly, pulmonary Ear11 transcripts, which are absent in naive mice, accumulate as a consequence of OVA-mediated TH2 inflammation in the lung. The induction of Ear11 expression is dependent on the presence of T cells, in particular, CD4+ T lymphocytes. This effect is likely the result of the elaboration of TH2 cytokine levels, because pulmonary instillation of interleukin-4 or interleukin-13 induces the accumulation of Ear11 transcripts in naive animals. This study demonstrates that despite an allergen-mediated pulmonary eosinophilia and earlier studies showing that Ears are constituents of eosinophil secondary granules, alveolar macrophages are a significant source of these RNases in lungs of OVA-treated mice.

SummaryPurposeIn a population‐based retrospective cohort of children with newly diagnosed epilepsy, to determine (1) what proportion meet criteria for early medical intractability, and (2) predictors of enduring intractability.MethodsChildren with newly diagnosed epilepsy between 1980 and 2009 while resident in Olmsted County, MN, and followed >36 months, were stratified into groups based on both early medical intractability (“apparent” medical intractability in the first 2 years) and enduring intractability (persisting intractability at final follow‐up or having undergone surgery for intractable epilepsy), and variables predicting these outcomes were evaluated.Key FindingsThree hundred eighty‐one children were included, representing 81% of our cohort with newly diagnosed epilepsy. Seventy five (19.7%) had early medical intractability, and predictors of this outcome on multivariable analysis were neuroimaging abnormality (risk ratio, 2.70; p = 0.0004), abnormal neurologic examination at diagnosis (risk ratio, 1.87; p = 0.015), and mode of onset (association was significant for focal vs. generalized onset [risk ratio, 0.25; p < 0.0001] but not unknown vs. generalized onset [p = 0.065]). After a median follow‐up of 11.7 years, 49% remained intractable, 8% had rare seizures (≤ every 6 months), and the remainder were seizure‐free. The only factor predicting enduring intractability on multivariable analysis was neuroimaging abnormality (risk ratio, 7.0; p = 0.0006).SignificanceAlthough a significant minority of children with early medical intractability ultimately achieved seizure control without surgery, those with an abnormal imaging study did poorly. For this subgroup, early surgical intervention is strongly advised to limit comorbidities of ongoing, intractable seizures. Conversely, a cautious approach is suggested for those with normal imaging, as most will remit with time.

Abstract We previously utilized a whole genome tiling array to identify novel transcripts which had altered expression in response to the genotoxic effects of the tobacco carcinogen NNK. Our goal was to identify novel long stress-induced non-coding transcripts that could play important regulatory roles within cells and be important targets of alteration during cancer formation. Utilizing very stringent criteria, we examined all transcriptionally active regions that had altered expression in response to NNK treatment and identified a group of 12 transcripts, termed long stress-induced noncoding trancripts (LSINCTs). These novel long non-coding transcripts are between 2 and 4 Kb in length and do not contain any open reading frames or have homology to any mRNAs. 11 LSINCTs are intergenically located and one LSINCT is intragenically located within the genome. We found LSINCTs to be more highly expressed in more proliferative normal human tissues and to be frequently over-expressed in a number of different cancers including breast cancer. We studied one of these transcripts in greater detail, LSINCT5. We found LSINCT5 to be a polyadenylated nuclear transcript of exactly 2,647 base pairs. It is highly over-expressed in both breast and ovarian cancers and knocking down its' expression in either a breast or ovarian cancer cell line resulted in decreased cellular proliferation. We further compared gene expression in the breast cancer cell line, MCF7, before and after knocking down LSINCT5 expression and identified a number of genes whose expression changed more than 2-fold in response. Two of the genes identified were the gene for the major paraspeckle protein PSPC1, and another long non-coding transcript NEAT1. Paraspeckles are irregularly shaped compartments found in the nucleus' interchromatin space. Paraspeckles are RNA-protein structures formed by the interaction of NEAT1 and members of the Drosophila Behavior Human Splicing family of proteins including PSPC1. Paraspeckles appear to be critical to the control of gene expression through the nuclear retention of certain species of RNA. It is though that paraspeckles and their components play a role in controlling gene expression during a number of different cellular processes including differentiation, viral infection and stress responses. Using RNA fluorescent in situ hybridization, we found LSINCT5 to co-localize with NEAT1 within the nucleus. Hence, this novel stress-induced transcript may be another important long non-coding transcript that is involved in the normal cellular function of the paraspeckles. Citation Format: David I. Smith, Silva M. Jessica. Long stress-induced non-coding transcripts (LSINCTs) and Cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A1.