Circulating Nucleated Red Blood Cells: An Updated Reference Interval.

CONTEXT.­: Nucleated red blood cells (nRBCs) are not identified in the peripheral blood in healthy individuals beyond the neonatal period. Their presence in children and adults is traditionally considered pathologic. Contemporary hematology analyzers measure nRBCs at very low levels compared to traditional manual morphometric methods. The original launch of the Sysmex XN analyzer in this study's clinical laboratory verified the previously used nRBC reference interval of 0.00 to 0.01 × 106/µL. However, nRBC results from apparently healthy patients were flagged as abnormal (high), subsequently causing patient anxiety and increased subspecialty referrals. OBJECTIVE.­: To determine whether current reference intervals (RIs) for nRBCs were clinically relevant. DESIGN.­: We performed a prospective analysis of 405 300 specimens from nonhospitalized individuals who received a complete blood count. Applying inclusion/exclusion criteria produced a total specimen pool of 66 498. RESULTS.­: Of the 66 498 samples with otherwise normal complete blood count results from healthy, nonhospitalized individuals, 338 showed results outside the previously established RI; 336 of 66 498 (0.5%) had nRBC results greater than 0.01 × 106/µL. Two samples had nRBC values greater than 0.10 ×106/µL. CONCLUSIONS.­: Based on statistical analysis of our results, we concluded that the upper limit of the RI could be updated from 0.01 × 106/µL to 0.10 × 106/µL. Increasing the upper limit of normal for the nRBC RI should decrease patient consternation from an abnormal laboratory value and significantly decrease costs through reducing unnecessary follow up care, and without causing patient harm.

Prospective Clinical Prognostication of Endometrial Carcinomas Based on Next-Generation Sequencing and Immunohistochemistry-Real-World Implementation and Results at a Tertiary Care Center.

Based on findings from The Cancer Genome Atlas and the Proactive Molecular Risk Classifier for Endometrial Cancer algorithm, endometrial carcinoma can now be stratified into 4 prognostically distinct subgroups based on molecular alterations and immunohistochemical (IHC) aberrations. In this study, we describe the de novo adoption and clinical reporting of prognostic subgroup classification based on next-generation sequencing (NGS) and IHC analyses of all endometrial carcinoma resections at a single institution, framed by the Exploration, Preparation, Implementation, and Sustainment model. Results from the first 13 months show 188 tumors underwent analysis by a combination of IHC and a medium-sized (56 analyzed genes) NGS-based assay. All cases were assigned as either POLE (POLE-mutated) (5.3%), mismatch repair deficient (27.7%), no specific molecular profile (45.7%), or p53 abnormal (21.3%) inclusive of multiple-classifier cases. NGS-based analysis revealed additional distinctions among the subgroups, including reduced levels of PI3K pathway activation in the p53 abnormal subgroup, an increased rate of CTNNB1 activating mutation in the no specific molecular profile subgroup, and lower TP53 mutation variant allele frequencies in POLE and mismatch repair deficient subgroups compared with the p53 abnormal subgroup. Overall, we describe the testing protocol, reporting, and results of a combination of NGS and IHC to prospectively prognosticate endometrial carcinomas at a single tertiary care center.

It Does Exist! Diagnosis and Management of Thyroid Carcinomas Originating in Struma Ovarii.

Thyroid carcinoma originating in struma ovarii comprises a small minority of all cases of struma ovarii. Given the rarity of this diagnosis, literature to guide evaluation and management is limited. The most common carcinoma originating from struma ovarii is papillary thyroid carcinoma. Treatment includes surgery, including a fertility sparing approach if disease is confined to the ovary, with consideration of total thyroidectomy and radioactive iodine ablation for high-risk pathologic features or disease spread beyond the ovary. This review discusses the histopathologic findings, molecular pathology, clinical implications and management, and prognosis of thyroid carcinomas originating in struma ovarii.

Adenocarcinoma of the uterine cervix, gastric-type (GAS): a review of the literature focused on pathology and multimodality imaging.

Adenocarcinoma of the uterine cervix, gastric-type (GAS) is a rare, well-differentiated subtype of HPV-independent endocervical adenocarcinoma. It classically arises in middle-aged women with symptoms, including profuse watery vaginal discharge and abnormal uterine bleeding. Given the rarity of this disease, misdiagnosis is common and prognosis remains poorly defined. Distinct pathology and imaging findings can aid in diagnosis. A literature review was performed to ascertain recurring pathologic and radiologic characteristics of GAS. Key pathologic features of GAS include cytologically benign appearing mucinous glands that infiltrate into the deep stroma and may demonstrate lymphovascular or perineural invasion. Multiple imaging modalities including transvaginal ultrasound, CT, and MRI may aid in diagnosis of GAS, which characteristically is seen as a multicystic mass with solid components. MRI in particular is the preferred imaging study because it offers the best chance of identifying a potential solid component, which is key to making the diagnosis of GAS and distinguishing it from other endocervical diseases processes. Careful attention to histopathologic and radiologic details, in conjunction with clinical correlation, is necessary to distinguish GAS from other multicystic cervical lesions.

Tryptophan catabolism in epithelial ovarian carcinoma.

Ovarian cancers are the most common cause of gynecological death, and the five-year survival rate for women diagnosed with epithelial ovarian carcinoma (EOC) remains extremely low at only 47%. Recent studies have highlighted the importance of the anti-tumor immune response in determining EOC clinical outcomes, and much research is currently being undertaken in an effort to reverse tumor immune evasion. One mechanism known to promote tumor immune evasion in multiple cancer types is tryptophan catabolism. Here we review the potential role of two rate-limiting enzymes that evolved separately to catabolize tryptophan, indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase 2 (TDO2), that may be active in ovarian cancers and result in the production of immune suppressive catabolites. Research to date has focused on IDO inhibitors, currently in clinical trials, but these therapies fail to inhibit TDO2. However, our mining of publically available data from clinical specimens suggest that TDO2 may also need to be targeted in ovarian cancer.

RTP801 Amplifies Nicotinamide Adenine Dinucleotide Phosphate Oxidase-4-Dependent Oxidative Stress Induced by Cigarette Smoke.

Tobacco smoke (TS) causes chronic obstructive pulmonary disease, including chronic bronchitis, emphysema, and asthma. Rtp801, an inhibitor of mechanistic target of rapamycin, is induced by oxidative stress triggered by TS. Its up-regulation drives lung susceptibility to TS injury by enhancing inflammation and alveolar destruction. We postulated that Rtp801 is not only increased by reactive oxygen species (ROS) in TS but also instrumental in creating a feedforward process leading to amplification of endogenous ROS generation. We used cigarette smoke extract (CSE) to model the effect of TS in wild-type (Wt) and knockout (KO-Rtp801) mouse lung fibroblasts (MLF). The production of superoxide anion in KO-Rtp801 MLF was lower than that in Rtp801 Wt cells after CSE treatment, and it was inhibited in Wt MLF by silencing nicotinamide adenine dinucleotide phosphate oxidase-4 (Nox4) expression with small interfering Nox4 RNA. We observed a cytoplasmic location of ROS formation by real-time redox changes using reduction-oxidation-sensitive green fluorescent protein profluorescent probes. Both the superoxide production and the increase in the cytoplasmic redox were inhibited by apocynin. Reduction in the activity of Sod and decreases in the expression of Sod2 and Gpx1 genes were associated with Rtp801 CSE induction. The ROS produced by Nox4 in conjunction with the decrease in cellular antioxidant enzymatic defenses may account for the observed cytoplasmic redox changes and cellular damage caused by TS.

Novosphingobium and its potential role in chronic obstructive pulmonary diseases: insights from microbiome studies.

Bacterial infection of lung airways underlies some of the main complications of COPD, significantly impacting disease progression and outcome. Colonization by bacteria may further synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, contributing to the characteristic airway inflammation and alveolar destruction of COPD. We sought to elucidate the presence and types of lung bacterial populations in different stages of COPD, aimed at revealing important insights into the pathobiology of the disease. Sequencing of the bacterial small subunit ribosomal RNA gene in 55 well-characterized clinical lung samples, revealed the presence of Novosphingobium spp. (>2% abundance) in lungs of patients with GOLD 3-GOLD 4 COPD, cystic fibrosis and a subset of control individuals. Novosphingobium-specific quantitative PCR was concordant with the sequence data and high levels of Novosphingobium spp. were quantifiable in advanced COPD, but not from other disease stages. Using a mouse model of subacute lung injury due to inhalation of cigarette smoke, bronchoalveolar lavage neutrophil and macrophage counts were significantly higher in mice challenged intratracheally with N. panipatense compared to control mice (p<0.01). Frequencies of neutrophils and macrophages in lung tissue were increased in mice challenged with N. panipatense at room air compared to controls. However, we did not observe an interaction between N. panipatense and subacute cigarette smoke exposure in the mouse. In conclusion, Novosphingobium spp. are present in more severe COPD disease, and increase inflammation in a mouse model of smoke exposure.

Rtp801 suppression of epithelial mTORC1 augments endotoxin-induced lung inflammation.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Pulmonary veins in the normal lung and pulmonary hypertension due to left heart disease.

Despite the importance of pulmonary veins in normal lung physiology and the pathobiology of pulmonary hypertension with left heart disease (PH-LHD), pulmonary veins remain largely understudied. Difficult to identify histologically, lung venous endothelium or smooth muscle cells display no unique characteristic functional and structural markers that distinguish them from pulmonary arteries. To address these challenges, we undertook a search for unique molecular markers in pulmonary veins. In addition, we addressed the expression pattern of a candidate molecular marker and analyzed the structural pattern of vascular remodeling of pulmonary veins in a rodent model of PH-LHD and in lung tissue of patients with PH-LHD obtained at time of placement on a left ventricular assist device. We detected urokinase plasminogen activator receptor (uPAR) expression preferentially in normal pulmonary veins of mice, rats, and human lungs. Expression of uPAR remained elevated in pulmonary veins of rats with PH-LHD; however, we also detected induction of uPAR expression in remodeled pulmonary arteries. These findings were validated in lungs of patients with PH-LHD. In selected patients with sequential lung biopsy at the time of removal of the left ventricular assist device, we present early data suggesting improvement in pulmonary hemodynamics and venous remodeling, indicating potential regression of venous remodeling in response to assist device treatment. Our data indicate that remodeling of pulmonary veins is an integral part of PH-LHD and that pulmonary veins share some key features present in remodeled yet not normotensive pulmonary arteries.

Heparanase mediates renal dysfunction during early sepsis in mice.

Heparanase, a heparan sulfate-specific glucuronidase, mediates the onset of pulmonary neutrophil adhesion and inflammatory lung injury during early sepsis. We hypothesized that glomerular heparanase is similarly activated during sepsis and contributes to septic acute kidney injury (AKI). We induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N-desulfated re-N-acetylated heparin [NAH]). Four hours after surgery, we collected serum and urine for measurement of renal function and systemic inflammation, invasively determined systemic hemodynamics, harvested kidneys for histology/protein/mRNA, and/or measured glomerular filtration by inulin clearance. CLP-treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular filtration, as indicated by a >twofold increase in blood urea nitrogen (BUN) and a >50% decrease in inulin clearance (P < 0.05) in comparison to sham mice. Administration of heparanase inhibitors 2 h prior to CLP attenuated sepsis-induced loss of glomerular filtration rate, demonstrating that heparanase activation contributes to early septic renal dysfunction. Glomerular heparanase activation was not associated with renal neutrophil influx or altered vascular permeability, in marked contrast to previously described effects of pulmonary heparanase on neutrophilic lung injury during sepsis. CLP induction of renal inflammatory gene (IL-6, TNF-α, IL-1ß) expression was attenuated by NAH pretreatment. While serum inflammatory indices (KC, IL-6, TNF-α, IL-1ß) were not impacted by NAH pretreatment, heparanase inhibition attenuated the CLP-induced increase in serum IL-10. These findings demonstrate that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via mechanisms disparate from heparanase-mediated lung injury.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

α7-Nicotinic acetylcholine receptor: role in early odor learning preference in mice.

Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7) in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP), an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5-18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor) for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21), mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits.

Typically developing and speech-impaired children's adherence to the sonority hypothesis.

During phonological development, children frequently produce consonant clusters as consonant singletons, a process commonly referred to as cluster reduction. The principles of sonority may provide a theoretical basis for explaining patterns of cluster reduction evident in children's speech. Two studies were conducted to investigate whether children's word-initial cluster reductions adhered to the sonority hypothesis. Study one involved 16 children with typically developing speech, and study two involved 40 children with impaired speech. The children's consonant cluster productions characterized by a cluster reduction were analysed. When both groups of participants reduced word-initial clusters to a target consonant, the sonority hypothesis was adhered to; but when the clusters were reduced to a non-target consonant, the sonority hypothesis was violated. Analysis of target and non-target reductions revealed that some reductions of the individual clusters, and those within specific cluster categories, adhered to the sonority hypothesis while others did not. In light of these findings, it is suggested that although sonority is a valuable concept, it may not account for all patterns of cluster reduction evident in children's speech.