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Background: The project was performed to determine if referrals to non-surgical providers after an initial presentation of symptomatic cholelithiasis are associated with a delay in surgical management., Methods: A single institution chart review of all adult patients who underwent a cholecystectomy from 2015 to 2019 was completed. Quantitative data was analyzed using independent t-tests., Results: Of 574 reviewed, 482 patients met criteria. Following initial presentation, 295 (61.2%) received a referral to surgery and 187 (38.8%) received follow up with a non-surgical provider. Those in the latter group had a significantly longer time from initial symptom presentation to surgical evaluation (65.7 days vs. 10.3 days, p ​< ​0.001) and cholecystectomy (102.0 days vs 39.1 days, p ​< ​0.001) when compared to the surgery referral group., Conclusions: This study demonstrated that cholecystectomy was significantly delayed for patients who had been referred to non-surgical providers after initial presentation, prolonging symptoms and increasing use of healthcare resources., Competing Interests: Declaration of competing interest This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Published by Elsevier Inc.)

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies., (© 2022 by The American Society of Hematology.)

SARS-CoV-2 infection, and resulting disease, COVID-19, has a high mortality amongst patients with haematological malignancies. Global vaccine rollouts have reduced hospitalisations and deaths, but vaccine efficacy in patients with haematological malignancies is known to be reduced. The UK-strategy offered a third, mRNA-based, vaccine as an extension to the primary course in these patients. The MARCH database is a retrospective observational study of serological responses in patients with blood disorders. Here we present data on 381 patients with haematological malignancies. By comparison with healthy controls, we report suboptimal responses following two primary vaccines, with significantly enhanced responses following the third primary dose. These responses however are heterogeneous and determined by haematological malignancy sub-type and therapy. We identify a group of patients with continued suboptimal vaccine responses who may benefit from additional doses, prophylactic extended half-life neutralising monoclonal therapies (nMAB) or prompt nMAB treatment in the event of SARS-CoV-2 infection., (© 2022. The Author(s).)

Patients with haematological malignancies have a high risk of severe infection and death from SARS-CoV-2. In this prospective observational study, we investigated the impact of cancer type, disease activity, and treatment in 877 unvaccinated UK patients with SARS-CoV-2 infection and active haematological cancer. The primary end-point was all-cause mortality. In a multivariate analysis adjusted for age, sex and comorbidities, the highest mortality was in patients with acute leukaemia [odds ratio (OR) = 1·73, 95% confidence interval (CI) 1·1-2·72, P = 0·017] and myeloma (OR 1·3, 95% CI 0·96-1·76, P = 0·08). Having uncontrolled cancer (newly diagnosed awaiting treatment as well as relapsed or progressive disease) was associated with increased mortality risk (OR = 2·45, 95% CI 1·09-5·5, P = 0·03), as was receiving second or beyond line of treatment (OR = 1·7, 95% CI 1·08-2·67, P = 0·023). We found no association between recent cytotoxic chemotherapy or anti-CD19/anti-CD20 treatment and increased risk of death within the limitations of the cohort size. Therefore, disease control is an important factor predicting mortality in the context of SARS-CoV-2 infection alongside the possible risks of therapies such as cytotoxic treatment or anti-CD19/anti-CD20 treatments., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

An understanding of adipocyte responsiveness to G-protein-coupled receptor-(GPCR) derived signals must take into consideration the role of membrane microenvironments; that individual sub-populations of proteins may vary significantly across different regions of the cell, and that cell differentiation alters those microenvironments. 3T3-L1 pre-adipocytes undergo a dramatic phenotypic transformation during differentiation into adipocytes, requiring the development of a transient primary cilium. We demonstrate that melanin-concentrating hormone (MCH) receptor 1, a GPCR that stimulates appetite, translocates to the transient primary cilium during early 3T3-L1 cell adipogenesis. Furthermore, we used RNA-Seq to investigate whether MCH signaling is influenced by its receptor localization and whether MCH can influence the transcriptome of early adipocyte development. We found that MCH signaling is sensitive to receptor localization to cilia, and this alters the adipogenic transcriptional program. Also, novel MCH signaling pathways in 3T3-L1 cells are identified, including those for circadian rhythm, the inflammatory response, and ciliary biogenesis. The presence of active MCH-signaling pathways in pre-adipocytes and the discovery that these pathways intersect with the early adipogenic program, among other newly-identified signaling pathways, suggests that the use of MCH receptor 1 antagonists for clinical interventions may have unintended consequences on adipose tissue development.

Background: The effects of epidural anesthesia in dogs undergoing cystoscopy are unknown., Objective: To investigate the effect of epidural analgesia on postcystoscopy pain in dogs., Animals: Twenty-six dogs undergoing routine cystoscopy for lower urinary tract disease., Methods: Prospective, randomized, blinded observational study. Dogs were assigned either to a treatment group that received epidural anesthesia (preservative free morphine sulfate, 0.09 mg/kg; 1% ropivacaine, 0.2 mg/kg; total volume delivered, 1 mL/4.5 kg of body weight to a maximum of 10 mL; n = 9) or to a nonepidural control group (n = 13). Vital signs were monitored for 24 hours, and sedation and pain scores, behavioral assessments, and presence or absence of complications was evaluated for 5 days postprocedure., Results: All dogs tolerated the epidural without complications. Four dogs were removed from the study because of status unblinding, lack of patient cooperation, or incomplete follow-up. No significant differences were noted in postprocedural pain scores in dogs that received epidural analgesia. Significant differences in postprocedural pain scores were noted in the nonepidural control group. No significant differences were noted in vital signs, behavioral assessments, or the proportion of dogs with a 50% increase in pain scores between the epidural and nonepidural groups., Conclusions and Clinical Importance: Epidural anesthesia was well-tolerated. Dogs not receiving the epidural had poor postprocedural pain control. A consistent benefit for the epidural vs nonepidural group could not be identified. Additional studies are required to better assess the impact and efficacy of epidural anesthesia for cystoscopic procedures., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive T-cell malignancy that arises in a proportion of individuals who are long-term carriers of human T-lymphotropic virus type 1. The median survival of aggressive subtypes is 8 to 10 months; with chemotherapy-based approaches, overall survival has remained largely unchanged in the ∼35 years since ATL was first described. Through the use of 4 representative case studies, we highlight advances in the biological understanding of ATL and the use of novel therapies such as mogamulizumab, as well as how they are best applied to different subtypes of ATL. We discuss the implementation of molecular methods that may guide diagnosis or treatment, although we accept that these are not universally available. In particular, we acknowledge discrepancies in treatment between different countries, reflecting current drug licensing and the difficulties in making treatment decisions in a rare disease, with limited high-quality clinical trial data., (© 2021 by The American Society of Hematology.)

Introduction: Chronic infection with human T-cell lymphotropic virus type-1 (HTLV-1) may result in aggressive adult T-cell leukaemia/lymphoma (ATL) in 4-6% carriers. The majority of this risk arises in carriers infected during infancy, and so each infant has ∼25% lifetime risk. Other risk factors include a family history of ATL. Antenatal HTLV-1 screening is not undertaken in the UK., Methods: Here we describe four cases of ATL diagnosed during pregnancy and describe strategies to minimise HTLV-1 transmission to neonates., Results/conclusion: These cases highlight undiagnosed HTLV-1 in pregnancy which allows ongoing mother to child vertical transmission and risk of future ATL. We recommend the UK National Screening Committee incorporate HTLV-1 serology into antenatal screening., (© 2020 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Adult T-cell leukemia/lymphoma (ATL) is an aggressive hematological malignancy caused by human T-cell leukemia virus type-1 (HTLV-1). ATL is preceded by decades of chronic HTLV-1 infection, and the tumors carry both somatic mutations and proviral DNA integrated into the tumor genome. In order to gain insight into the oncogenic process, we used targeted sequencing to track the evolution of the malignant clone in 6 individuals, 2 to 10 years before the diagnosis of ATL. Clones of premalignant HTLV-1-infected cells bearing known driver mutations were detected in the blood up to 10 years before individuals developed acute and lymphoma subtype ATL. Six months before diagnosis, the total number and variant allele fraction of mutations increased in the blood. Peripheral blood mononuclear cells from premalignant cases (1 year prediagnosis) had significantly higher mutational burden in genes frequently mutated in ATL than did high-risk, age-matched HTLV-1 carriers who remained ATL-free after a median of 10 years of follow-up. These data show that HTLV-1-infected T-cell clones carrying key oncogenic driver mutations can be detected in cases of ATL years before the onset of symptoms. Early detection of such mutations may enable earlier and more effective intervention to prevent the development of ATL., (© 2020 by The American Society of Hematology.)

Background: Syringomyelia (SM) is a debilitating condition in the cavalier King Charles spaniel (CKCS) that results in neuropathic pain and diminished quality of life. Von Frey aesthesiometry (VFA) is a method of mechanical quantitative sensory testing that provides an objective sensory threshold (ST) value and can be used to quantify neuropathic pain (NP) and monitor response to therapy. The utility of VFA has been previously established in client-owned dogs with acute spinal cord injury but the technique has not been evaluated in dogs with SM. The goal of this study was to evaluate ST, as determined by VFA, in dogs with and without SM, to assess the utility of VFA in quantifying NP in SM-affected dogs. We hypothesized the SM-affected CKCS would have lower ST values, consistent with hyperesthesia, when compared to control CKCS. Additionally, we hypothesized that ST values in SM-affected dogs would be inversely correlated with syrinx size on MRI and with owner-derived clinical sign scores., Results: ST values for the thoracic and pelvic limbs differed significantly between the SM-affected and control CKCS (p = 0.027; p = 0.0396 respectively). Median ST value (range) for the thoracic limbs was 184.1 g (120.9-552) for control dogs, and 139.9 g (52.6-250.9) for SM-affected dogs. The median ST value (range) for the pelvic limbs was 164.9 g (100.8-260.3) in control dogs and 129.8 g (57.95-168.4) in SM-affected dogs. The ST values in SM-affected dogs did not correlate with syrinx height on MRI (r = 0.314; p = 0.137). Owner-reported clinical sign scores showed an inverse correlation with pelvic limb ST values, where dogs with lower ST values (hyperesthesia) were reported by their owners to display more frequent and severe clinical signs (r = - 0.657; p = 0.022)., Conclusion: ST values were lower in SM-affected CKCS compared to control dogs, suggesting the presence of neuropathic pain. Dogs with lower ST pelvic limb values were perceived by their owners to have more severe clinical signs classically associated with SM. Our results suggest that VFA might offer quantitative assessment of neuropathic pain in SM-affected dogs and could be useful for monitoring response to therapy in future clinical studies.

Electronic von Frey Aesthesiometry (VFA) has been previously reported as a useful method of mechanical quantitative sensory testing (QST) for evaluating neuropathic pain in dogs. Intraobserver agreement has been shown to be good to excellent; however, interobserver agreement has not been evaluated and is vital to the use of this technique in multicenter veterinary clinical trials in neuropathic pain. The goal of this study was to evaluate the interobserver agreement of sensory thresholds obtained using electronic VFA in a group of normal small breed dogs. Twenty healthy dogs (<20 kg) were recruited from the general practice population at the Ohio State University Veterinary Medical Center. Three clinically experienced yet QST novice evaluators used an electronic von Frey device to measure mechanical sensory threshold (ST) after a standardised training session conducted by an expert evaluator. Each dog was assessed by all three evaluators on the same day with both evaluator and limb test order randomised and testing sessions separated by 5 min. Mean ST values were averaged for all four limbs to produce a single value per dog for comparison between evaluators. Agreement between evaluators was determined using the intra-class correlation coefficient (ICC; two-way model for consistency, single measures). ICC across all three evaluators was 0.48, indicating moderate agreement. Moderate interobserver agreement is not sufficient to support the use of this technique in multi-center clinical trials, and our results underscore the importance of using a single evaluator for this QST technique until better agreement can be demonstrated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Adult T-cell leukemia-lymphoma (ATL) is a distinct mature T-cell malignancy caused by chronic infection with human T-lymphotropic virus type 1 with diverse clinical features and prognosis. ATL remains a challenging disease as a result of its diverse clinical features, multidrug resistance of malignant cells, frequent large tumor burden, hypercalcemia, and/or frequent opportunistic infection. In 2009, we published a consensus report to define prognostic factors, clinical subclassifications, treatment strategies, and response criteria. The 2009 consensus report has become the standard reference for clinical trials in ATL and a guide for clinical management. Since the last consensus there has been progress in the understanding of the molecular pathophysiology of ATL and risk-adapted treatment approaches., Methods: Reflecting these advances, ATL researchers and clinicians joined together at the 18th International Conference on Human Retrovirology-Human T-Lymphotropic Virus and Related Retroviruses-in Tokyo, Japan, March, 2017, to review evidence for current clinical practice and to update the consensus with a new focus on the subtype classification of cutaneous ATL, CNS lesions in aggressive ATL, management of elderly or transplantation-ineligible patients, and treatment strategies that incorporate up-front allogeneic hematopoietic stem-cell transplantation and novel agents., Results: As a result of lower-quality clinical evidence, a best practice approach was adopted and consensus statements agreed on by coauthors (> 90% agreement)., Conclusion: This expert consensus highlights the need for additional clinical trials to develop novel standard therapies for the treatment of ATL.

Objectives: The aim of this study was to compile an overview of the clinical features of intracranial complication of otitis media/interna (OMI) in cats managed across five veterinary referral hospitals. Of additional interest were culture results that could inform empirical antibiotic selection, as well as outcome with both medical and surgical management., Methods: A retrospective medical record review was conducted at five veterinary referral practices to identify cats with a diagnosis of intracranial complication secondary to OMI between 2009 and 2017. Clinical features, diagnostic findings, treatment and outcome were recorded., Results: At total of 19 cats were identified. Sixty-three percent had no previous history of ear infection. Otoscopic examination was normal in 47% of cases. The most common bacterial isolate was Pasteurella multocida, which was identified in 24% of cases. Outcome was successful for 83% of cats managed with ventral bulla osteotomy (VBO) and in 66% of cats managed without surgical intervention., Conclusions and Relevance: Clinical suspicion of intracranial complications of OMI should remain high in cats with central vestibular disease even if otoscopic examination is normal. Antibiotic selection should be based on a culture and sensitivity; however, initial antibiotic therapy should include broad-spectrum coverage with special consideration for P multocida. Cats with intracranial complications of OMI can have a good outcome with either surgical or medical management and prospective studies are needed to assess the role of VBO in enhancing recovery.

Globally, > 5-10 million people are estimated to be infected with Human T-lymphotropic virus type 1 (HTLV-1), of whom ~ 5% develop adult T-cell leukemia/lymphoma (ATL). Despite advances in chemotherapy, overall survival (OS) has not improved in the 35 years since HTLV-1 was first described. In Europe/USA, combination treatment with zidovudine and interferon-α (ZDV/IFN-α) has substantially changed the management of patients with the leukemic subtypes of ATL (acute or unfavorable chronic ATL) and is under clinical trial evaluation in Japan. However, there is only a single published report of long-term clinical remission on discontinuing ZDV/IFN-α therapy and the optimal duration of treatment is unknown. Anecdotal cases where therapy is discontinued due to side effects or compliance have been associated with rapid disease relapse, and it has been widely accepted that the majority of patients will require life-long therapy. The development of molecular methods to quantify minimal residual disease is essential to potentially guide therapy for individual patients. Here, for the first time, we report molecular evidence that supports long-term clinical remission in a patient who was previously treated with ZDV/IFN-α for 5 years, and who has now been off all therapy for over 6 years.

Many nursing theorists claim caring as the aspect of nursing that differentiates it from other health care professions. Newman et al defined the focus of nursing as "the study of caring in the human health experience," yet there is little consensus among nurses about the validity of this statement. The purpose of this article is to explore the concept of caring as it has been defined by various nursing scholars and to determine whether the focus of nursing knowledge development can appropriately be defined as "the study of caring in the human health experience."

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease., Competing Interests: The authors have declared that no competing interests exist.

Cystic lesions of the vertebral column and spinal cord are important differential diagnoses in dogs with signs of spinal cord disease. Synovial cysts are commonly associated with degenerative joint disease and usually affect the cervical and lumbosacral regions. Arachnoid diverticulum (previously known as cyst) is seen in the cervical region of large breed dogs and thoracolumbar region of small breed dogs. This article reviews the causes, diagnosis, and treatment of these and other, less common, cystic lesions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis., Results: HTLV-1 antibodies were first detected 16-39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16-23 and increased by 2-3 log by day 38-45 with a peak proviral doubling time of 1.4 days, after which steady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation., Conclusions: In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early anti-HTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread.

Purpose of Review: To discuss current understanding of the mechanisms of human T-lymphotropic virus type-1 (HTLV-1) tumorigenesis and current and potential treatment strategies for adult T-cell leukaemia/lymphoma (ATL), an aggressive malignant disease of CD4 cells caused by HTLV-1., Recent Findings: Treatment of the aggressive subtypes of ATL remains inadequate, with little improvement in overall survival in the 30 years since HTLV-1 was discovered. Detailed analysis of the clonal expansion of HTLV-1 has provided new insight into pathogenesis. Most HTLV-1-infected cells, including ATL, express CCR4 which can be targeted. Reports of antitumour effects with allogeneic bone marrow transplantation provide a rationale for novel immunotherapy approaches. Progress has been made in the indolent subtypes of ATL with the use of 'antiviral' therapies., Summary: ATL has poor prognosis. There is a major, urgent, unmet clinical need to identify HTLV carriers who will develop ATL to develop biomarkers of transforming disease and disease progression and to provide novel treatment approaches within the context of clinical trials. Several strategies now include putative or actual antiviral therapy. Potentially, the risk of ATL would be reduced by eliminating some or all infected clones. HTLV-1 infection, and hence ATL, can be prevented by antenatal HTLV-1 screening.

Adult T-cell leukemia/lymphoma (ATL) occurs in ∼5% of human T-lymphotropic virus type 1 (HTLV-1)-infected individuals and is conventionally thought to be a monoclonal disease in which a single HTLV-1(+) T-cell clone progressively outcompetes others and undergoes malignant transformation. Here, using a sensitive high-throughput method, we quantified clonality in 197 ATL cases, identified genomic characteristics of the proviral integration sites in malignant and nonmalignant clones, and investigated the proviral features (genomic structure and 5' long terminal repeat methylation) that determine its capacity to express the HTLV-1 oncoprotein Tax. Of the dominant, presumed malignant clones, 91% contained a single provirus. The genomic characteristics of the integration sites in the ATL clones resembled those of the frequent low-abundance clones (present in both ATL cases and carriers) and not those of the intermediate-abundance clones observed in 24% of ATL cases, suggesting that oligoclonal proliferation per se does not cause malignant transformation. Gene ontology analysis revealed an association in 6% of cases between ATL and integration near host genes in 3 functional categories, including genes previously implicated in hematologic malignancies. In all cases of HTLV-1 infection, regardless of ATL, there was evidence of preferential survival of the provirus in vivo in acrocentric chromosomes (13, 14, 15, 21, and 22)., (© 2014 by The American Society of Hematology.)

Human T lymphotropic virus type 1 (HTLV-1) causes a range of chronic inflammatory diseases and an aggressive malignancy of T lymphocytes known as adult T-cell leukaemia/lymphoma (ATLL). A cardinal feature of HTLV-1 infection is the presence of expanded clones of HTLV-1-infected T cells, which may persist for decades. A high viral burden (proviral load) is associated with both the inflammatory and malignant diseases caused by HTLV-1, and it has been believed that the oligoclonal expansion of infected cells predisposes to these diseases. However, it is not understood what regulates the clonality of HTLV-1 in vivo, that is, the number and abundance of HTLV-1-infected T cell clones. We review recent advances in the understanding of HTLV-1 infection and disease that have come from high-throughput quantification and analysis of HTLV-1 clonality in natural infection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The regulation of appetite is complex, though our understanding of the process is improving. The potential role for the melanin-concentrating hormone (MCH) signaling pathway in the treatment of obesity is being explored by many. It was hypothesized that internalization of MCH receptors would act to potently desensitize cells to MCH. Despite potent desensitization of ERK signaling by MCH in BHK-570 cells, we were unable to observe MCH-mediated internalization of MCH receptor 1 (MCHR1) by fluorescence microscopy. A more quantitative approach using a cell-based ELISA indicated only 15% of receptors internalized, which is much lower than that reported in the literature. When β-arrestins were overexpressed in our system, removal of receptors from the cell surface was facilitated and signaling to a leptin promoter was diminished, suggesting that internalization of MCHR1 is sensitive to cellular β-arrestin levels. A dominant-negative GRK construct completely inhibited loss of receptors from the cell surface in response to MCH, suggesting that the internalization observed is phosphorylation-dependent. Since desensitization of MCH-mediated ERK signaling did not correlate with significant loss of MCHR1 from the cell surface, we hypothesize that in this model system regulation of MCH signaling may be the result of segregation of receptors from signaling components at the plasma membrane.

Human T lymphotropic virus type 1 (HTLV-1) appears to persist in the chronic phase of infection by driving oligoclonal proliferation of infected T cells. Our recent high-throughput sequencing study revealed a large number (often > 10(4)) of distinct proviral integration sites of HTLV-1 in each host that is greatly in excess of previous estimates. Here we use the highly sensitive, quantitative high-throughput sequencing protocol to show that circulating HTLV-1(+) clones in natural infection each contain a single integrated proviral copy. We conclude that a typical host possesses a large number of distinct HTLV-1-infected T-cell clones.

Adipose tissue develops from differentiating preadipocytes that expand and migrate. 3T3-L1 preadipocytes respond to melanin-concentrating hormone (MCH) by increasing leptin production. Here, we investigate whether MCH elicits remodeling of the actin cytoskeleton and whether this translates into altered migratory capacity of these cells. Incubation with MCH resulted in a loss of actin stress fibers accompanied by a change in morphology from a stretched-out fibroblast to a rounded cell. PMC-3881-PI, a MCH receptor 1 antagonist blocked the effect, confirming this receptor is solely responsible for MCH-mediated actin rearrangements. Both a pharmacological activator and inhibitor of phospholipase C were used to demonstrate this molecule's importance to the signaling pathway. Finally, MCH was shown to facilitate preadipocyte migration into a scratch wound, revealing a previously unknown role for MCH in the regulation of cellular migration. We conclude that MCH could influence the expansion of adipose tissue through its ability to enhance preadipocyte migration., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X)(5-6)F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the beta2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.

The melanin-concentrating hormone receptor (MCHR) 1 is a G protein-coupled receptor involved in the regulation of appetite and energy expenditure in mammals. Here, we show that MCHR1 partitions to lipid rafts in stably expressing Chinese hamster ovary cells. In addition to co-fractionating with lipid rafts containing caveolin-1 on sucrose gradients, caveolin-1 was present in MCHR1 immunoprecipitates, suggesting that MCHR1 complexes with caveolae. The cholesterol-depleting drug methyl-beta-cyclodextrin impaired MCH-mediated ERK signaling. These data suggest that a functional interaction between MCHR1 and caveolin-1 in lipid rafts exists and provide a basis for further biochemical studies to understand the significance on MCH-mediated signal transduction events.

Vascular encephalopathy is a commonly encountered clinical complication of infectious endocarditis in humans, but it has been infrequently reported in dogs. A series of four dogs with bacterial endocarditis that subsequently developed acute onset of neurological deficits is described. Clinical signs, diagnostic test results, and outcomes for each case are presented and compared with the human clinical syndrome.

Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [(3)H][N(3)-methyl-His(2)]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75-80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [(3)H][N(3)-methyl-His(2)]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.

To study the effect of agonist on the TRH (thyrotrophin-releasing hormone) receptor protein, an epitope-tagged receptor was stably expressed in HEK-293 cells (human embryonic kidney 293 cells) and receptor levels were measured by immunoblotting. TRH caused a 5-25-fold increase in receptor protein during 48 h, which was half-maximal at 1 nM and was slowly reversible after hormone withdrawal. Chlordiazepoxide, an inverse agonist, had no effect. TRH up-regulation was mimicked by phorbol ester and blocked by the protein kinase C inhibitor GF109203X in combination with thapsigargin, which prevents a calcium response. TRH and phorbol ester increased the density of immunoreactive receptors localized at the cell surface and [3H]MeTRH (where MeTRH stands for [N3-methyl-His]TRH) binding. TRH also increased the concentration of a truncated, internalization-defective receptor. Analysis of cell lines stably expressing TRH receptors fused to the green fluorescent protein on a fluorescence-activated cell sorter showed that TRH and phorbol ester caused 2.7- and 6.8-fold increases in fusion protein expression respectively. TRH receptor up-regulation was only partially accounted for by changes in receptor mRNA, which increased 1.7-fold. TRH caused a small increase in receptor concentration in the presence of cycloheximide, actinomycin D or MG132. In contrast with the results obtained with the TRH receptor, agonist decreased the concentration of stably expressed b2-adrenergic receptors. These results show that TRH increases receptor concentration by a complex mechanism that requires signal transduction but not receptor endocytosis.

Diseases of the ear often cause signs of neurologic dysfunction because of damage of peripheral nervous system structures associated with the middle and inner ear. Vestibular dysfunction, facial paralysis, Horner's syndrome, and hearing deficits are the most common neurologic deficits that accompany middle and inner ear disease. Differentiating these signs from disease of the central nervous system is crucial for an accurate diagnosis and prognosis but can be difficult. Understanding the normal anatomy of the ear and its association with the brain is crucial to interpretation of the neurologic examination. This article reviews neurologic dysfunction commonly associated with diseases of the ear and differentiating these signs from central disease.

A 15-month-old male Maine Coon Cat presented with persistent auricular discharge and progressive head tilt, ataxia, and loss of blink on the right side. Using computed tomography a hyperattenuating, contrast-enhancing material within a thickened right tympanic bulla and contrast enhancement of the adjacent cerebellum were identified. Marked suppurative inflammation was identified on cerebrospinal fluid analysis with no growth on bacterial culture. Ventral bulla osteotomy was performed to remove a soft tissue mass, and an inflammatory polyp with chronic severe suppurative inflammation was confirmed using histology. Staphylococcus auricularis was grown on aerobic culture and Fusobacterium necrophorum and Peptostreptococcus anaerobius were grown on anaerobic culture. The cat was treated for 10 weeks with amoxicillin/clavulinic acid and metronidazole. Dramatic improvement in body weight, appetite, energy level, balance, and resolution of right-sided facial paralysis were noted, but the cat retained a head tilt.

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.

This study compared derived arterial flow waveforms, extracted from pulse oximeter waveforms, with Doppler flow waveforms. Fingertip pulse oximeter waveforms and radial artery Doppler flow waveforms were measured simultaneously in volunteers. The pulse oximeter waveforms were processed to extract the arterial flow waveforms and these were compared with the measured Doppler waveforms. They were very similar.

A method is described which allows an approximation to the arterial flow waveform to be derived from a pulse oximeter waveform. The observed pulse oximeter waveform is the sum of arterial inflow and venous outflow. These components are separated mathematically. Subtraction of the venous outflow reveals the underlying arterial flow waveform. The assumptions on which the method is based are stated explicitly and discussed.

This double-blind, randomized trial compared the onset of sedation with two patient-controlled sedation regimens, allowing a maximum of 16 or 25 mg min-1 propofol. Forty fit young patients presenting for elective surgery were asked to try to put themselves to sleep using the system. Onset times of sedative effect, slurred speech and amnesia were recorded. All patients achieved satisfactory sedation, and none became oversedated. Patients receiving 16 mg min-1 propofol were not reliably sedated within 5 min and took significantly longer to develop slurred speech and amnesia (P < 0.01 for both). We conclude that this maximum infusion rate does not produce amnesia or sedation rapidly enough to be clinically useful. A maximum infusion rate of 25 mg min-1 allowed rapid sedation in all patients without oversedation and may be an acceptable compromise between efficacy and safety.

We have investigated the safety of a previously described method of patient-controlled sedation in 100 healthy patients presenting for elective surgery. The device used 0.33-ml boluses of propofol (10 mg.ml-1) infused over 6 s with no lockout. All patients attempted to put themselves to sleep by pressing a button on the hand-held device. The oxygen saturation, heart rate, sedation score and airway patency were noted every minute. When the patient stopped pressing the button, the anaesthetist took over the handset and continued pressing the button until the patient became unresponsive. The system allowed rapid sedation. Only five patients were still anxious after 2 min. After 3 min, no patient was still anxious and more than 70 patients had slurred speech. When they stopped pressing the button, 11 patients were judged oversedated, of whom two were unresponsive. One patient's oxygen saturation decreased transiently to 84%: no other patient's oxygen saturation decreased below 90%. There were no other significant changes. We conclude that the system studied works well but carries too high a risk of oversedation for unsupervised use.

The degree of rebreathing in Mapleson A, C and D breathing systems for sinusoidal and exponential flow waveforms is analysed mathematically. The effects of altering the I:E ratio and of introducing an expiratory pause are investigated. The results for sinusoidal waveforms closely resemble those for a square wave. Exponential flow waveforms produce results similar to triangular flow waveforms. The Mapleson A system is always the most efficient. The Mapleson C system is efficient when the I:E ratio is 1:1, becoming less efficient with longer expiration and very inefficient with an expiratory pause. The Mapleson D system becomes efficient when the expiratory pause is long.

A physical lung model simulating spontaneous respiration was used to investigate the influence of the respiratory pattern on the efficiency of the Mapleson A, C and D breathing systems. It is shown that the Mapleson A system is always the most efficient breathing system and that its performance is relatively independent of the respiratory pattern. When the expiratory pause is minimal, the Mapleson C system is almost as efficient as the Mapleson A, but becomes ever less efficient as the expiratory pause increases. The Mapleson D system is very inefficient when the expiratory pause is short. With a longer expiratory pause, this system's efficiency approaches that of the Mapleson A. The experimental results are compared with predictions generated by a mathematical model. There is good agreement between the two, validating the mathematics used.

A theoretical analysis is presented of the Mapleson A, C and D breathing systems when used in spontaneous respiration. The influence of the respiratory pattern is explained diagrammatically. Simple equations are derived, predicting the fresh gas flow required to prevent rebreathing with different respiratory patterns. Further equations allow the degree of rebreathing caused by inadequate fresh gas flow to be quantified. These are used to examine the effects of different respiratory patterns on the efficiency of the three systems. It is demonstrated that the single most important determinant of efficiency is the duration of the expiratory pause. The nature of the inspiratory and expiratory waveforms is less important and the I:E ratio far less important. The analysis suggests that the Mapleson A system will always be the most efficient of the three systems. The Mapleson C system will be efficient if inspiration is long and the expiratory pause is minimal. The Mapleson D system will be efficient if the expiratory pause is sufficiently long.

The performance of breathing systems is tested by means of physical lung models. We describe a new lung model, allowing direct measurement of the dead space/tidal volume ratios produced by any breathing system at different fresh gas flows. The model allows a range of different patterns of spontaneous respiration to be studied. Although rebreathing is measured by capnography, the design ensures that the results are unaffected by changes in carbon dioxide inflow, or even calibration drift in the capnograph.

A laboratory study of a new circle system is presented. The inspiratory limb encloses a coaxial tube, delivering fresh gas close to the patient, immediately upstream of the inspiratory one-way valve. The system is more efficient than the non-coaxial system and allows the expiratory valve to be situated conveniently at the canister end of the expiratory limb. A disadvantage of low flow anaesthesia is the sluggish response of the system to changes in vapour or oxygen concentrations in the fresh gas flow. This is markedly improved by the new system. The advantages of the system are largely independent of respiratory pattern.

The hypothesis that anaesthetic uptake during maintenance of anaesthesia is related to cardiac output was tested on 21 patients undergoing cardiac surgery. Using a computer-controlled closed breathing system, enflurane was administered to maintain an end-expired concentration of 1%. Cardiac output was measured by thermodilution using a pulmonary artery catheter. A clear qualitative but not quantitative relationship was demonstrated. Changes in anaesthetic requirements at a constant end-expired concentration are a better guide to changes in cardiac output than changes in end-expired carbon dioxide with constant ventilation in patients undergoing cardiac surgery.

The introduction of balloon-tipped pulmonary artery flotation catheters revolutionised monitoring of the cardiovascular system and the management of seriously ill patients. Advances in design allow direct measurement of cardiac output and continuous mixed venous oxygen saturation, while intracardiac electrocardiogram monitoring and passage of pacing wires is now possible. The technique of insertion must be meticulous as must the method of obtaining central venous, pulmonary artery, pulmonary artery wedge and pulmonary capillary wedge pressures. Sources of error are highlighted as are methods to avoid and detect them. Correct interpretation of the data is essential for proper patient management and must be taken in light of information obtained by other methods. Complications of insertion are numerous and potentially serious or even lethal. It is mandatory that the information obtained by insertion of a pulmonary artery catheter be used to optimize patient management.