Search

Killer whales ( Orcinus orca ) are experiencing increasing environmental pressures, with some ecotypes being identified as endangered, and the development and validation of non-invasive health assessment tools is critical for assessing the well-being of individuals within these endangered populations. Infrared thermography of the blowhole is a non-contact method of temperature measurement that was recently investigated in killer whales in managed care. Two male killer whales presenting with clinical signs at separate institutions had veterinary clinical health assessments performed, which included infrared thermography of the blowhole as well as concurrent rectal temperature measurement. The current case report is aimed at describing the clinical use of infrared thermography of the blowhole as a method to detect elevated body temperature in two killer whales. Both animals exhibited blowhole temperatures above the previously reported values (36.4 °C and 37.6 °C; the mean in healthy whales is reported to be 34.21 ± 1.47 °C) with concurrently elevated rectal temperatures, as well as clinicopathologic findings consistent with a systemic inflammatory response (e.g., neutrophilia, increased fibrinogen and erythrocyte sedimentation rate, hypoferritinemia). Following veterinary intervention, both animals' blowhole and rectal temperatures returned to baseline. Infrared thermography of the blowhole represents a promising tool for the identification of pyrexic animals and with further investigation may be considered as part of conservation health assessments for threatened free-ranging populations.

Case Description: A 19-year-old male bottlenose dolphin (Tursiops truncatus) presented with inappetence and avoidant behavior., Clinical Findings: Ultrasound revealed a large-volume left-sided pleural effusion, which was consistent with chronic nonchylous lymphatic effusion and mild chronic hemorrhage by cytology. Computed tomography identified ipsilateral rib fractures, atelectasis, nodular pleuritis, marginal lymph node enlargement, and suspected dilation of the thoracic duct and internal thoracic veins. Fifteen lipids were significantly higher in serum of the dolphin as compared with controls (n = 3) using nontargeted lipidomics., Treatment and Outcome: A series of thoracentesis procedures were performed. Follow-up CT demonstrated marked reduction in pleural effusion with persistence of thoracic duct dilation and mass-like areas of pleural thickening. Ultrasonographic resolution of pleural effusion occurred 14 months after presentation; however, recrudescence was noted 5 months later. Over a total of 24 months, 21.52 L of pleural effusion was removed. Despite the presence of pleural effusion, the patient was clinically stable during this time and quality of life was considered good on the basis of continuous animal welfare evaluations. Humane euthanasia was elected following acute clinical decline 27 months after initial diagnosis. Necropsy confirmed severe pleural effusion, chronic severe pleural fibrosis with chronic hemorrhage, and mediastinal fibrosis with entrapped lymph nodes and thymic tissue., Clinical Relevance: Pleuritis and effusion were suspected sequelae of previous rib fractures. To our knowledge, this is the first report of nonchylous lymphatic pleural effusion with repeated pleural drainage and diagnostic imaging for clinical management in a bottlenose dolphin.

Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFβ type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFβ family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L’s function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2 + stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2 + PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2 + PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells., Competing Interests: JR, XL, AS, VY, YK, BW, MF, SH, AG, SH, EL, AP, AS, PM, CA No competing interests declared, RN Reviewing editor, eLife, (© 2021, Russell et al.)

Background: The expression of RET in the developing enteric nervous system (ENS) suggests that RET may contribute to adult intestinal function. ENS cholinergic nerves play a critical role in the control of colonic function through the release of acetylcholine (ACh). In the current study, we hypothesized that a RET-mediated mechanism may regulate colonic ion transport and motility through modulation of cholinergic nerves., Methods: The effect of RET inhibition on active ion transport was assessed electrophysiologically in rat colonic tissue mounted in Ussing chambers via measurements of short circuit current (Isc) upon electrical field stimulation (EFS) or pharmacologically with cholinergic agonists utilizing a gastrointestinal (GI)-restricted RET inhibitor. We assessed the effect of the RET inhibitor on propulsive motility via quantification of fecal pellet output (FPO) induced by the acetylcholinesterase inhibitor neostigmine., Key Results: We found that enteric ganglia co-expressed RET and choline acetyltransferase (ChAT) transcripts. In vitro, the RET kinase inhibitor GSK3179106 attenuated the mean increase in Isc induced by either EFS or carbachol but not bethanechol. In vivo, GSK3179106 significantly reduced the prokinetic effect of neostigmine., Conclusion and Inferences: Our findings provide evidence that RET-mediated mechanisms regulate colonic function by maintaining cholinergic neuronal function and enabling ACh-evoked chloride secretion and motility. We suggest that modulating the cholinergic control of the colon via a RET inhibitor may represent a novel target for the treatment of intestinal disorders associated with increased secretion and accelerated GI transit such as irritable bowel syndrome with diarrhea (IBS-D)., (© 2018 John Wiley & Sons Ltd.)

SOX2 positive pituitary stem cells (PSCs) are specified embryonically and persist throughout life, giving rise to all pituitary endocrine lineages. We have previously shown the activation of the STK/LATS/YAP/TAZ signalling cascade in the developing and postnatal mammalian pituitary. Here, we investigate the function of this pathway during pituitary development and in the regulation of the SOX2 cell compartment. Through loss- and gain-of-function genetic approaches, we reveal that restricting YAP/TAZ activation during development is essential for normal organ size and specification from SOX2+ PSCs. Postnatal deletion of LATS kinases and subsequent upregulation of YAP/TAZ leads to uncontrolled clonal expansion of the SOX2+ PSCs and disruption of their differentiation, causing the formation of non-secreting, aggressive pituitary tumours. In contrast, sustained expression of YAP alone results in expansion of SOX2+ PSCs capable of differentiation and devoid of tumourigenic potential. Our findings identify the LATS/YAP/TAZ signalling cascade as an essential component of PSC regulation in normal pituitary physiology and tumourigenesis., Competing Interests: EL, AS, JR, PX, TJ, RJ, ST, SB, CA No competing interests declared, (© 2019, Lodge et al.)

Abdominal pain represents a significant complaint in patients with irritable bowel syndrome (IBS). While the etiology of IBS is incompletely understood, prior exposure to gastrointestinal inflammation or psychologic stress is frequently associated with the development of symptoms. Inflammation or stress-induced expression of growth factors or cytokines may contribute to the pathophysiology of IBS. Here, we aimed to investigate the therapeutic potential of inhibiting the receptor of glial cell line-derived neurotrophic factor, rearranged during transfection (RET), in experimental models of inflammation and stress-induced visceral hypersensitivity resembling IBS sequelae. In RET-cyan fluorescent protein [(CFP) Ret CFP/+ ] mice, thoracic and lumbosacral dorsal root ganglia were shown to express RET, which colocalized with calcitonin gene-related peptide. To understand the role of RET in visceral nociception, we employed GSK3179106 as a potent, selective, and gut-restricted RET kinase inhibitor. Colonic hyperalgesia, quantified as exaggerated visceromotor response to graded pressures (0-60 mm Hg) of isobaric colorectal distension (CRD), was produced in multiple rat models induced 1) by colonic irritation, 2) following acute colonic inflammation, 3) by adulthood stress, and 4) by early life stress. In all the rat models, RET inhibition with GSK3179106 attenuated the number of abdominal contractions induced by CRD. Our findings identify a role for RET in visceral nociception. Inhibition of RET kinase with a potent, selective, and gut-restricted small molecule may represent a novel therapeutic strategy for the treatment of IBS through the attenuation of post-inflammatory and stress-induced visceral hypersensitivity., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

As a central regulator of major physiological processes, the pituitary gland is a highly dynamic organ, capable of responding to hormonal demand and hypothalamic influence, through adapting secretion as well as remodelling cell numbers among its seven populations of differentiated cells. Stem cells of the pituitary have been shown to actively generate new cells during postnatal development but remain mostly quiescent during adulthood, where they persist as a long-lived population. Despite a significant body of research characterising attributes of anterior pituitary stem cells, the regulation of this population is poorly understood. A better grasp on the signalling mechanisms influencing stem proliferation and cell fate decisions can impact on our future treatments of pituitary gland disorders such as organ failure and pituitary tumours, which can disrupt endocrine homeostasis with life-long consequences. This minireview addresses the current methodologies aiming to understand better the attributes of pituitary stem cells and the normal regulation of this population in the organ, and discusses putative future avenues to manipulate pituitary stem cells during disease states or regenerative medicine approaches., (© 2018 S. Karger AG, Basel.)

Orthopedic dogma states that external fixator stiffness is improved by placing 1 pin close to the fracture and 1 as distant as possible ("near-far"). This fixator construct is thought to be less expensive than placing pins a shorter distance apart and using "pin-bar" clamps that attach pins to outriggers. The authors therefore hypothesized that the near-far construct is stiffer and less expensive. They compared mechanical stiffness and costs of near-far and pin-bar constructs commonly used for temporary external fixation of femoral shaft fractures. Their testing model simulated femoral shaft fractures in damage control situations. Fourth-generation synthetic femora (n=18) were used. The near-far construct had 2 pins that were 106 mm apart, placed 25 mm from the gap on each side of the fracture. The pin-bar construct pins were 55 mm apart, placed 40 mm from the gap. Mechanical testing was performed on a material test system machine. Stiffness was determined in the linear portion of the load-displacement curve for both constructs in 4 modes: axial compression, torsional loading, frontal plane 3-point bending, and sagittal plane 3-point bending. Costs were determined from a 2012 price guide. Compared with the near-far construct, the pin-bar construct had stiffness increased by 58% in axial compression (P<.05) and by 52% in torsional loading (P<.05). The pin-bar construct increased cost by 11%. In contrast to the authors' hypothesis and existing orthopedic dogma, the near-far construct was less stiff than the pin-bar construct and was similarly priced. Use of the pin-bar construct is mechanically and economically reasonable. [Orthopedics. 2017; 40(2):e238-e241.]., (Copyright 2016, SLACK Incorporated.)

Background: Allograft tendons are frequently used for ligament reconstruction about the knee, but they entail availability and cost challenges. The identification of other tissues that demonstrate equivalent performance to preferred tendons would improve limitations. Hypothesis/Purpose: We compared the biomechanical properties of 4 soft tissue allograft tendons: tibialis anterior (TA), tibialis posterior (TP), peroneus longus (PL), and semitendinosus (ST). We hypothesized that allograft properties would be similar when standardized by the looped diameter., Study Design: Controlled laboratory study., Methods: This study consisted of 2 arms evaluating large and small looped-diameter grafts: experiment A consisted of TA, TP, and PL tendons (n = 47 each) with larger looped diameters of 9.0 to 9.5 mm, and experiment B consisted of TA, TP, PL, and ST tendons (n = 53 each) with smaller looped diameters of 7.0 to 7.5 mm. Each specimen underwent mechanical testing to measure the modulus of elasticity (E), ultimate tensile force (UTF), maximal elongation at failure, ultimate tensile stress (UTS), and ultimate tensile strain (UTε)., Results: Experiment A: No significant differences were noted among tendons for UTF, maximal elongation at failure, and UTϵ. UTS was significantly higher for the PL (54 MPa) compared with the TA (44 MPa) and TP (43 MPa) tendons. E was significantly higher for the PL (501 MPa) compared with the TP (416 MPa) tendons. Equivalence testing showed that the TP and PL tendon properties were equivalent or superior to those of the TA tendons for all outcomes. Experiment B: All groups exhibited a similar E. UTF was again highest in the PL tendons (2294 N) but was significantly different from only the ST tendons (1915 N). UTϵ was significantly higher for the ST (0.22) compared with the TA (0.19) and TP (0.19) tendons. Equivalence testing showed that the TA, TP, and PL tendon properties were equivalent or superior to those of the ST tendons., Conclusion: Compared with TA tendons, TP and PL tendons of a given looped diameter exhibited noninferior initial biomechanical strength and stiffness characteristics. ST tendons were mostly similar to TA tendons but exhibited a significantly higher elongation/UTϵ and smaller cross-sectional area. For smaller looped-diameter grafts, all tissues were noninferior to ST tendons. In contrast to previous findings, PL tendons proved to be equally strong., Clinical Relevance: The results of this study should encourage surgeons to use these soft tissue allografts interchangeably, which is important as the number of ligament reconstructions performed with allografts continues to rise.

The pituitary gland is a primary endocrine organ that controls major physiological processes. Abnormal development or homeostatic disruptions can lead to human disorders such as hypopituitarism or tumors. Multiple signaling pathways, including WNT, BMP, FGF, and SHH regulate pituitary development but the role of the Hippo-YAP1/TAZ cascade is currently unknown. In multiple tissues, the Hippo kinase cascade underlies neoplasias; it influences organ size through the regulation of proliferation and apoptosis, and has roles in determining stem cell potential. We have used a sensitive mRNA in situ hybridization method (RNAscope) to determine the expression patterns of the Hippo pathway components during mouse pituitary development. We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active. We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state. Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.

During arthroscopic Bankart repair, penetration of suture anchors through the far cortex can compromise the initial biomechanical characteristics of anchor stability and repair integrity. This study compared the placement of suture anchors through a low anterior-inferior rotator interval portal (AI) vs a trans-subscapularis portal to evaluate the rate of anchor perforation as well as biomechanical strength. Ten matched pairs of cadaveric shoulders were randomized to an AI or a trans-subscapularis portal for placement of suture anchors at the 3 o'clock and 5:30 positions. The following measurements were obtained: (1) distance from the portal to the cephalic vein; (2) presence and length of anchor penetration through the inferior glenoid; and (3) ultimate failure strength of the anchors. The distance from the portal to the cephalic vein was significantly greater with the AI vs the trans-subscapularis portal across all specimens (29.9 vs 11.2 mm, P<.05). The rate of anchor penetration was significantly increased in the AI group vs the trans-subscapularis group at the 5:30 position (60% vs 10%, P=.014) but not at the 3 o'clock position (P=.33). Mean pullout strength of the anchors at the 5:30 position trended higher in the trans-subscapularis group, but the difference was not significant (132.8 vs 112.6 N, P=.18). The cephalic vein is closer to the trans-subscapularis portal than to the AI, but is at a safe distance. Both the rate and the degree of glenoid suture anchor penetration were lower with the trans-subscapularis portal compared with the AI at the 5:30 position. Placing anchors through the trans-subscapularis portal provides a safe alternative method, with improved positioning of the inferiormost anchor compared with the traditional AI., (Copyright 2016, SLACK Incorporated.)

Background: Multiple rib fractures cause significant pain and potential for chest wall instability. Despite an emerging trend of surgical management of flail chest injuries, there are no studies examining the effect of rib fracture fixation on respiratory function., Objectives: Using a novel full thorax human cadaveric breathing model, we sought to explore the effect of flail chest injury and subsequent rib fracture fixation on respiratory outcomes., Patients and Methods: We used five fresh human cadavers to generate negative breathing models in the left thorax to mimic physiologic respiration. Inspiratory volumes and peak flows were measured using a flow meter for all three chest wall states: intact chest, left-sided flail chest (segmental fractures of ribs 3 - 7), and post-fracture open reduction and internal fixation (ORIF) of the chest wall with a pre-contoured rib specific plate fixation system., Results: A wide variation in the mean inspiratory volumes and peak flows were measured between specimens; however, the effect of a flail chest wall and the subsequent internal fixation of the unstable rib fractures was consistent across all samples. Compared to the intact chest wall, the inspiratory volume decreased by 40 ± 19% in the flail chest model (P = 0.04). Open reduction and internal fixation of the flail chest returned the inspiratory volume to 130 ± 71% of the intact chest volumes (P = 0.68). A similar 35 ± 19% decrease in peak flows was seen in the flail chest (P = 0.007) and this returned to 125 ± 71% of the intact chest following ORIF (P = 0.62)., Conclusions: Negative pressure inspiration is significantly impaired by an unstable chest wall. Restoring mechanical stability of the fractured ribs improves respiratory outcomes similar to baseline values.

Neural stem/progenitor cells (NSCs) in the hippocampus produce new neurons throughout adult life. NSCs are maintained in a state of reversible quiescence and the failure to maintain the quiescent state can result in the premature depletion of the stem cell pool. The epigenetic mechanisms that maintain this quiescent state have not been identified. Using an inducible knockout mouse model, we show that the chromatin remodeling factor chromodomain-helicase-DNA-binding protein 7 (CHD7) is essential for maintaining NSC quiescence. CHD7 inactivation in adult NSCs results in a loss of stem cell quiescence in the hippocampus, a transient increase in cell divisions, followed by a significant decline in neurogenesis. This loss of NSC quiescence is associated with the premature loss of NSCs in middle-aged mice. We find that CHD7 represses the transcription of several positive regulators of cell cycle progression and is required for full induction of the Notch target gene Hes5 in quiescent NSCs. These findings directly link CHD7 to pathways involved in NSC quiescence and identify the first chromatin-remodeling factor with a role in NSC quiescence and maintenance. As CHD7 haplo-insufficiency is associated with a range of cognitive disabilities in CHARGE syndrome, our observations may have implications for understanding the basis of these deficits., (© 2014 AlphaMed Press.)

Objectives: Little is known about the mechanical properties of internal anterior fixators (known as INFIX), which have been proposed as subcutaneous alternatives to traditional anterior external fixators for pelvic ring disruptions. We hypothesised that INFIX has superior biomechanical performance compared with traditional external fixators because the distance from the bar to the bone is reduced., Methods: Using a commercially available synthetic bone model, 15 unstable pelvic ring injuries were simulated by excising the pubic bone through the bilateral superior and inferior rami anteriorly and the sacrum through the bilateral sacral foramen posteriorly. Three test groups were established: (1) traditional supra-acetabular external fixation, (2) INFIX with polyaxial screws, (3) INFIX with monaxial screws. Load was applied, simulating lateral compression force. Outcome measure was construct stiffness., Results: The traditional external fixator constructs had an average stiffness of 6.21 N/mm ± 0.40 standard deviation (SD). INFIX with monaxial screws was 23% stiffer than the traditional external fixator (mean stiffness, 7.66 N/mm ± 0.86 SD; p = .01). INFIX with polyaxial screws was 26% less stiff than INFIX with monaxial screws (mean stiffness, 5.69 N/mm ± 1.24 SD; p = .05). No significant difference was noted between polyaxial INFIX and external fixators (mean stiffness, 6.21 N/mm ± 0.40 SD; p=.65)., Conclusions: The performance of INFIX depends on the type of screw used, with monaxial screws providing significantly more stiffness than polyaxial screws. Despite the mechanical advantage of being closer to the bone, polyaxial INFIX was not stiffer than traditional external fixation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We compared the mechanical benefits and costs of 3 strategies that are commonly used to increase knee-spanning external fixator stiffness (resistance to deformation): double stacking, cross-linking, and use of an oblique pin. At our academic trauma centre and biomechanical testing laboratory, we used ultra-high-molecular-weight polyethylene bone models and commercially available external fixator components to simulate knee-spanning external fixation. The models were tested in anterior-posterior bending, medial-lateral bending, axial compression, and torsion. We recorded the construct stiffness for each strategy in all loading modes and assessed a secondary outcome of cost per 10% increase in stiffness. Double stacking significantly increased construct stiffness under anterior-posterior bending (109%), medial-lateral bending (22%), axial compression (150%), and torsion (41%) (p<0.05). Use of an oblique pin significantly increased stiffness under torsion (25%) (p<0.006). Cross-linking significantly increased stiffness only under torsion (29%) (p<0.002). Double stacking increased costs by 84%, cross-linking by 28%, and use of an oblique pin by 15% relative to a standard fixator. All 3 strategies increased stiffness under torsion to varying degrees, but only double stacking increased stiffness in all 4 testing modalities (p<0.05). Double stacking is most effective in increasing resistance to bending, particularly under anterior-posterior bending and axial compression, but requires a relatively high cost increase. Clinicians can use these data to help guide the most cost-effective strategy to increase construct stiffness based on the plane in which stiffness is needed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The authors' objective was to determine the effects of bar diameter on the stiffness and cost of a knee-spanning external fixator. The authors studied 2 versions of an external fixator with a difference in bar diameter (small bars, 8-mm diameter; large bars, 11-mm diameter). Fixators were tested using frame dimensions and a synthetic fracture model appropriate for a tibial plateau fracture. Five configurations of each fixator were tested: standard, cross-link, oblique pin, double stack, and super construct. The construct stiffness of each configuration (n=60) was measured in anterior-posterior bending, medial-lateral bending, axial torsion, and axial compression. Cost analysis allowed for calculation of the stiffness per unit cost. In the large bar group, an increase in construct stiffness was noted for all constructs and testing modes. Magnitude of stiffness increase ranged from 24% to 224% (P<.05 in all cases), depending on the configuration and loading mode. Increase in stiffness was so large that double-stack small bars performed similarly to standard construct large bars. Considering that the frame components have similar costs, the double-stack small bar fixator results in a 66% increase in cost for the same stiffness provided by the standard large bar. Bar diameter seems to have a large effect on knee-spanning external fixators. The authors observed an increase in stiffness of up to 191% under anterior-posterior bending despite an increase in bar size of only 37.5%. This finding might allow clinicians to use less expensive frames constructed of larger bars without sacrificing construct stiffness., (Copyright 2014, SLACK Incorporated.)

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.

An accurate and robust electroencephalogram (EEG) source localization algorithm would be a definite asset for the surgical treatment of patients with epilepsy. Due to the underdetermined nature of the EEG inverse problem, a variety of algorithms with unique constraints and assumptions are applied to select the current dipole source distribution that best accounts for the scalp recordings. We investigated four algorithms: two non-adaptive algorithms: the minimum norm and LORETA as well as two adaptive algorithms: the Borgiotti-Kaplan and eigenspace projection beamformers. Compared over a range of SNR values and single source locations, we found that the eigenspace projection beamformer exhibited superior localizing capabilities compared to the other three algorithms while minimizing source current dispersion. The size of the data window required to accurately localize using the adaptive beamformers was also investigated to improve algorithm efficiency and minimize stationary source assumptions.

RET/PTC3 (RP3) is an oncogenic fusion protein which is frequently expressed in papillary thyroid carcinomas and has been detected in thyroid tissue from patients diagnosed with Hashimoto's thyroiditis. The constitutive activation of the tyrosine kinase domain in the carboxyl-terminal end of RP3 induces signaling pathways within thyrocytes and causes cellular transformation. One of the signaling pathways activated in RP3-expressing cells involves the activity of the transcription factor NF-kappaB and the production of downstream targets including GM-CSF and macrophage chemotactic protein 1. These factors are known to be immunostimulatory, making RP3 a molecular adjuvant and potentially promoting tissue-specific immunity. However compelling, these in vitro data do not reliably predict gene function in vivo or the cumulative effects of time-dependent processes such as angiogenesis, inflammation, or the influence of genetic background. To address these issues, we analyzed the production of proinflammatory mediators in mouse thyroid organs and demonstrate consistency with in vitro studies performed previously that Il1alpha, Il1beta, Il6, and Tnfalpha and the enzyme Cox2 are produced by RP3-transgenic thyroid tissue, but absent from nontransgenic thyroids. Furthermore, we find that that the genetic background of the host is important in the observed RP3-induced inflammation and tumor progression. These findings provide support for the notion that oncogene-induced cytokine secretion is important for the development and progression of thyroid carcinomas in genetically permissive hosts.

Disorders associated with cutaneous vasculitis include numerous well-described etiologies. Primary cutaneous vasculitis limits discussion to primary leukocytoclastic vasculitis, essential mixed cryoglobulinemia, urticarial vasculitis, Henoch-Schönlein purpura, and erythema elevatum diutinum. Although the therapeutics for these disorders are based on limited data, we attempt to construct a consensus opinion on the management of primary cutaneous vasculitis. Therapy of primary cutaneous vasculitis is indicated for symptomatic or systemic involvement, because cutaneous small vessel vasculitis is frequently a self-limited, single episodic disease. Conservative, symptomatic treatment includes leg elevation, warming, antihistamines, and nonsteroidal anti-inflammatory drugs. For mild recurrent disease, colchicine, dapsone, and prednisone are first-choice agents. Systemic or severe cutaneous disease requires more potent immunosuppression (eg, prednisone, azathioprine, or mycophenolate mofetil). Plasmapheresis/plasma exchange and intravenous immunoglobulin are viable considerations for refractory disease, but are cumbersome and expensive modalities. There is insufficient evidence to advocate the use of new biological or monoclonal antibody therapies in primary cutaneous vasculitis.

Objectives: Cyclooxygenases (COX) are enzymes that catalyze the conversion of arachidonic acid to prostaglandins. COX-2, unlike the constitutively expressed COX-1, is an inducible enzyme upregulated during cell proliferation and inflammation. More recently, COX-2 has been implicated in the development of numerous types of epithelial cancers. In addition, COX-2 is highly expressed in several inflammatory diseases. Because of its dual role in inflammation and cancer, we were interested in determining if COX-2 plays a role in the development of human thyroid carcinoma and Hashimoto's thyroiditis, an autoimmune condition frequently associated with thyroid malignancy., Materials and Methods: Twenty paraffin-embedded human tissue specimens, including normal, inflammatory, and neoplastic thyroid sections, were analyzed by immunohistochemical staining for expression of human COX-2. In addition, COX-2 protein expression was verified by Western blot in two specimens., Results: Immunohistochemical staining confirmed the presence of COX-2 in thyroid epithelial neoplasms, including papillary and follicular carcinomas. Moreover, COX-2 expression was observed in patients with Hashimoto's thyroiditis. COX-2 expression, however, was not observed in normal thyroid tissue, multinodular goiter, or anaplastic carcinoma., Conclusions: We have shown that cyclooxygenase-2 is expressed in thyroid carcinoma and thyroid epithelium from patients with Hashimoto's thyroiditis but not in normal thyroid. The expression of COX-2 in both of these thyroid pathologies may provide a basis for the relationship between carcinogenesis and autoimmunity.

Cancers develop and progress via activation of oncogenes and loss of tumor suppressor genes, a progression that can be recapitulated through cross breeding mouse strains harboring genetic mutations. To define the role of RET/PTC3, p53 and Fhit in thyroid carcinogenesis, we intercrossed RET/PTC3 transgenics with p53-/- mice. This new strain, RET/PTC3p53-/-, succumb to rapidly growing and strikingly large multilobed thyroid tumors containing mixtures of both well and poorly differentiated, highly proliferative follicular epithelial cells. Interestingly, transplanted tumors from RET/PTC3p53-/- mice grew in SCID but not syngeneic immunocompetent mice indicating that these advanced tumors were immunogenic. RET/PTC3 protein expression was reduced to undetectable levels in tumors of older mice suggesting that the continued elevated expression of RET/PTC3 may not be necessary for tumor progression. Similarly, expression of Fhit protein was reduced in early tumors and undetected in older tumors irrespective of tumor histopathology. In contrast to RET/PTC3p53-/- mice, RET/PTC3Fhit-/- mice did not develop advanced thyroid carcinomas. These studies support a model of human thyroid cancer whereby thyroid epithelium expresses RET/PTC3 protein at early stages of tumor development, followed by the reduction of RET/PTC3 and loss of p53 function with progressive reduction of Fhit protein expression coincident with malignant progression.

Genetic analysis of human papillary thyroid carcinomas (PTC) has revealed unique chromosomal translocations that form oncogenic fusion proteins and promote thyroid tumorigenesis in up to 60% of tumors examined. Although, the majority of thyroid specific translocations involve the growth factor receptor c-RET, variant rearrangements of the receptor for nerve growth factor, NTRK1 have also been described. One such translocation, TRK-T1, forms a fusion protein composed of the carboxyl terminal tyrosine kinase domain of NTRK1 and the amino terminal portion of TPR (Translocated Promoter Region). To determine if TRK-T1 expression can cause thyroid cancer in vivo, we developed transgenic mice that express the human TRK-T1 fusion protein in the thyroid. Immunohistochemical analysis of TRK-T1 transgenic mouse thyroids revealed TRK-T1 staining within the thyroid follicular epithelium. In contrast to nontransgenic littermates, 54% of transgenic mice developed thyroid abnormalities that included follicular hyperplasia and papillary carcinoma. Furthermore, all transgenic mice examined greater than 7 months of age developed thyroid hyperplasia and/or carcinoma. These data support the conclusion that TRK-T1 is oncogenic in vivo and contributes to the neoplastic transformation of the thyroid.

The paper describes a new, powerful, patented piece of sonic equipment that operates in the low sonic frequency range, at 104 Hz, with nominal power of 75 kW. It has been tested for both physical and chemical sonic effects. A large-scale industrial application is described--the conditioning and pacification of ash from utility coal combustion by fluidized bed combustion. A variety of additional applications is suggested, most of which have been briefly tested.

Innate immunity provides an ever-present or rapidly inducible initial defense against microbial infection. Among the effector molecules of this defense in many species are broad-spectrum antimicrobial peptides. Tracheal antimicrobial peptide (TAP) was the first discovered member of the beta-defensin family of mammalian antimicrobial peptides. TAP is expressed in the ciliated epithelium of the bovine trachea, and its mRNA levels are dramatically increased upon stimulation with bacteria or bacterial lipopolysaccharide (LPS). We report here that this induction by LPS is regulated at the level of transcription. Furthermore, the transfection of reporter gene constructs into tracheal epithelial cells indicates that DNA sequences in the 5' flanking region of the TAP gene, within 324 nucleotides of the transcription start site, are responsible in part for mediating gene induction. This region includes consensus binding sites for NF-kappaB and nuclear factor interleukin-6 (NF IL-6) transcription factors. Gel mobility shift assays indicate that LPS induces NF-kappaB binding activity in the nuclei of these cells, while NF IL-6 binding activity is constitutively present. The gene encoding human beta-defensin 2, a human homologue of TAP with similar inducible expression patterns in the airway, was cloned and found to have conserved NF-kappaB and NF IL-6 consensus binding sites in its 5' flanking region. Previous studies of antimicrobial peptides from insects indicated that their induction by infectious microbes and microbial products also occurs via activation of NF-kappaB-like and NF IL-6-like transcription factors. Together, these observations indicate that a strategy for the induction of peptide-based antimicrobial innate immunity is conserved among evolutionarily diverse organisms.

The CDR3 region of the TCR beta-chain of a CD4+, Th1, Vbeta2+ encephalitogenic T cell clone was used as an idiotypic marker to track the location of the clone in vivo. cDNA prepared from the spinal cord, thymus, lymph nodes, spleen, and liver of the recipients at various stages of EAE was amplified using Vbeta2 and Cbeta-region primers, and the products immobilized. The membrane was probed with a 32P-labeled oligonucleotide complementary to the CDR3 region of the T cell clone. The probe reacted strongly with products from the spinal cord, spleen and liver and less strongly with products from lymph nodes and thymus of mice with acute EAE. The signal was greatly diminished in the spinal cord and other tissues during recovery from acute disease and reappeared in the spinal cord at each relapse.

A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, beta-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with beta-defensin-related sequence [corrected] related to those of these alpha-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel beta-defensin, which we designate enteric beta-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized beta-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other beta-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other beta-defensin mRNAs were cloned. Most of these clones encoded previously characterized beta-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-beta-defensin. Northern blot evidence supported that all of these other beta-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these beta-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of beta-defensin expression in enteric tissue and that the mRNA encoding a major beta-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that beta-defensins may contribute to host defense of enteric mucosa.

Mammals continually confront microbes at mucosal surfaces. A current model suggests that epithelial cells contribute to defense at these sites, in part through the production of broad-spectrum antibiotic peptides. Previous studies have shown that invertebrates can mount a host defense response characterized by the induction in epithelia] cells of a variety of antibiotic proteins and peptides when they are challenged with microorganisms, bacterial cell wall/membrane components, or traumatic injury [Boman, H.G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126J. However, factors that govern the expression of similar defense molecules in mammalian epithelial cells are poorly understood. Here, a 13-fold induction of the endogenous gene encoding tracheal antimicrobial peptide was found to characterize a host response of tracheal epithelia] cells (TECs) exposed to bacterial lipopolysaccharide (LPS). Northern blot data indicated that TECs express CD14, a well-characterized LPS-binding protein known to mediate many LPS responses. A monoclonal antibody to CD14 blocked the observed tracheal antimicrobial peptide induction by LPS under serum-free conditions. Together the data support that CD14 of epithelial cell origin mediates the LPS induction of an antibiotic peptide gene in TECs, providing evidence for the active participation of epithelial cells in the host's local defense response to bacteria. Furthermore, the data allude to a conservation of this host response in evolution and suggest that a similar inducible pathway of host defense is prevalent at mucosal surfaces of mammals.

Peptides with potent broad-spectrum antibiotic activity have been identified in many animal species. Recent investigations have demonstrated that epithelial cells are a site of antibiotic peptide expression, suggesting that these peptides contribute to host defense at mucosal surfaces. Expression of tracheal antimicrobial peptide (TAP), a member of the beta-defensin family of peptides, is inducible in cultured tracheal epithelial cells (TEC) upon challenge with bacterial lipopolysaccharide (LPS) (G. Diamond, J.P. Russell, and C.L. Bevins, Proc. Natl. Acad. Sci. USA, in press). In this study, an anchored reverse transcriptase PCR strategy was used to determine if TAP was the sole beta-defensin isoform expressed upon stimulation of the cells with LPS. In addition to TAP, a second class of cDNA clones which encoded lingual antimicrobial peptide (LAP), a beta-defensin peptide recently isolated from a different mucosal site, the bovine tongue, was identified (B.S. Schonwetter, E.D. Stolzenberg, and M. Zasloff, Science 267:1645-1648, 1995). Northern (RNA) blot analysis demonstrated in vivo expression of LAP mRNA in tracheal mucosa. Levels of LAP mRNA were higher in cultured TEC challenged with either LPS or tumor necrosis factor alpha than in control cells. Thus, a response of TEC exposed to inflammatory mediators is induction of antibiotic-encoding genes, including both TAP and LAP. This work complements the in vivo studies of Schonwetter et al. (cited above), which showed elevated levels of LAP mRNA in squamous epithelial cells of the tongue near sites of tissue injury and inflammation, by suggesting possible mediators of the in vivo observation. Together these lines of investigations support the hypothesis that inducible expression of endogenous antibiotic peptides by inflammatory mediators characterizes local defense of mammalian mucosal surfaces.

Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5'-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19 24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.

The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.