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Salivary gland amyloidosis is an uncommon diagnosis. Most studies have focused on minor salivary gland biopsies as a surrogate site for diagnosing systemic amyloidosis, while only few studies have investigated major salivary gland amyloidosis. We retrospectively identified 57 major and minor salivary gland amyloidosis cases typed using a proteomics-based method between 2010 and 2022. Frequency of amyloid types, clinicopathologic features, and distribution patterns of amyloid deposits were assessed. The indication for salivary gland biopsy/resection (known in 34 cases) included suspected amyloidosis (N = 14; 41.2%), lesion/mass (N = 12; 35.3%), swelling/enlargement (N = 5; 14.7%), and rule out Sjogren syndrome (N = 3; 8.8%). Concurrent pathology was reported in 16 cases, and included chronic sialadenitis (N = 11), extranodal marginal zone lymphoma (N = 3), plasma cell neoplasm (N = 1), and pleomorphic adenoma (N = 1). We identified 3 types of amyloidosis: immunoglobulin light chain/AL (N = 47; 82.5%); immunoglobulin heavy chain/AH (N = 1; 1.8%), and transthyretin/ATTR (N = 9; 15.8%). The patterns of amyloid deposits (assessed in 35 cases) included: 1) Perivascular and/or periductal distribution (N = 18; 51.4%); 2) Mass formation (N = 9; 25.7%); 3) Stromal micronodule formation (N = 7; 20.0%); and 4) Diffuse interstitial involvement (N = 1; 2.9%). We also identified one case of AL amyloidosis localized to the major salivary gland, where only 6 other cases with adequate staging workup to exclude systemic amyloidosis were previously reported. In conclusion, salivary gland amyloidosis is an uncommon diagnosis but may be underrecognized due to low index of suspicion. Most cases of salivary gland amyloidosis are AL type, but a minority are ATTR. Therefore, proteomics-based typing remains essential for treatment and prognosis., Competing Interests: Declaration of competing interest April Chiu is a consultant with AstraZeneca. None for all other authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Context.—: The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities., Objective.—: To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003-2022), primarily utilizing G-banded karyograms., Design.—: A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated., Results.—: A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication., Conclusions.—: This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin + colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Adult intussusception is rare, highly associated with a malignant lead point, and often requires emergent surgical management. We report the case of a 44-year-old male who presented with generalized abdominal pain and was found to have early ileocolic intussusception secondary to a large ileocecal mass. Biopsies of the mass and an enlarged cardiophrenic lymph node, as well as pleural fluid cytology were all consistent with Burkitt lymphoma (BL). Curiously, the patient's abdominal exam was reassuring, and the intussusception and malignant bowel obstruction resolved over 36 hours with conservative management alone. With a Burkitt lymphoma international prognostic index (BL-IPI) score of 2, the patient proceeded to treatment with combination chemoimmunotherapy and attained a complete response after four cycles. There was no bowel perforation or recurrent intussusception throughout treatment. Thus, this report marks the first reported case of adult BL-associated intussusception to resolve with non-invasive management and establishes a precedent for conservative management in select patients., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this paper.

Purpose of Review: The treatment paradigm of chronic lymphocytic leukemia (CLL) has dramatically changed with the advent of novel targeted agents over the past decade. Richter transformation (RT), or the development of an aggressive lymphoma from a background of CLL, is a well-recognized complication of CLL and carries significantly poor clinical outcomes. Here, we provide an update on current diagnostics, prognostication, and contemporary treatment of RT., Recent Findings: Several genetic, biologic, and laboratory markers have been proposed as candidate risk factors for the development of RT. Although a diagnosis of RT is typically suspected based on clinical and laboratory findings, tissue biopsy is essential for histopathologic confirmation of diagnosis. The standard of care for RT treatment at this time remains chemoimmunotherapy with the goal of proceeding to allogeneic stem cell transplantation in eligible patients. Several newer treatment modalities are being studied for use in the management of RT, including small molecules, immunotherapy, bispecific antibodies, and chimeric antigen receptor T-cell (CAR-T) therapy. The management of patients with RT remains a challenge. Ongoing trials show enormous promise for newer classes of therapy in RT, with the hope being that these agents can synergize, and perhaps supersede, the current standard of care in the near future., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

T-lymphocytes are prevalent in the tumor microenvironment of follicular lymphoma (FL). However, the phenotype of T-cells may vary, and the prevalence of certain T-cell subsets may influence tumor biology and patient survival. We therefore analyzed a cohort of 82 FL patients using CyTOF to determine whether specific T-cell phenotypes were associated with distinct tumor microenvironments and patient outcome. We identified four immune subgroups with differing T-cell phenotypes and the prevalence of certain T-cell subsets was associated with patient survival. Patients with increased T cells with early differentiation stage tended to have a significantly better survival than patients with increased T-cells of late differentiation stage. Specifically, CD57 + T FH cells, with a late-stage differentiation phenotype, were significantly more abundant in FL patients who had early disease progression and therefore correlated with an inferior survival. Single cell analysis (CITE-seq) revealed that CD57 + T FH cells exhibited a substantially different transcriptome from CD57 - T FH cells with upregulation of inflammatory pathways, evidence of immune exhaustion and susceptibility to apoptosis. Taken together, our results show that different tumor microenvironments among FL patients are associated with variable T-cell phenotypes and an increased prevalence of CD57 + T FH cells is associated with poor patient survival., (© 2023. Springer Nature Limited.)

The role of the tumor microenvironment (TME) and intratumoral T cells in splenic marginal zone lymphoma (sMZL) is largely unknown. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of T FH cells varied greatly in sMZL, ICOS + T FH cells were more abundant in sMZL than rSP, and T FH cells positively correlated with increased numbers of memory B cells. T reg cell analysis revealed that TIGIT + T reg cells are enriched in sMZL and correlate with suppression of T H 17 and T H 22 cells. Intratumoral CD8 + T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1 low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data suggest that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM., (© 2022 by The American Society of Hematology.)

Composite classic Hodgkin lymphoma and follicular lymphoma (CHLFL) is a rare and poorly characterized entity. Herein, we report the clinicopathologic features of 22 cases of CHLFL from 3 institutions and we assess 27 additional cases reported in the literature. In our cohort (n=22), patients with CHLFL had a median age of 61 years and an equal male to female incidence. Most cases (95%) arose de novo with the remaining patients having a history of non-Hodgkin lymphoma. CHLFL always involved lymph nodes (100%) and most cases (95%) revealed 2 distinct areas separately diagnostic for CHL and FL. The CHL component represented a variable proportion of the overall neoplasm (5% to 90%) and was either mixed cellularity (82%) or nodular sclerosis (18%) type. The Hodgkin/Reed-Sternberg cells expressed CD30 (100%), PAX5 (100%), CD15 (62%), BCL6 (47%), BCL2 (29%), and EBER (25%), in a polymorphous inflammatory background typical of CHL. The FL component was low-grade in 55%, grade 3A in 36%, and grade 3B in 9% of cases. All 3 cases investigated by cytogenetic methods for a clonal relationship between the CHL and FL components were clonally related. These clinicopathologic features of our cohort are similar to those of cases reported in the literature. The 5-year overall survival in combined patients with CHLFL (n=49) was 48%, comparable to CHL but worse than FL in the elderly. In summary, CHLFL is a rare entity that most often occurs in older adults, involves lymph nodes, and most commonly presents de novo. In the small number of cases assessed, the CHL and FL components are usually clonally related suggesting that the CHL and FL components may share a common progenitor B-cell, likely a mutated germinal center B-cell., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To assess the prevalence of distress and burnout in otolaryngology trainees, including associations with relevant sociodemographic and professional factors, and to compare these results with those of attending otolaryngologists., Study Design: A cross-sectional survey of trainees and attending physicians., Setting: Twelve academic otolaryngology programs., Methods: Distress and burnout were measured with the Expanded Physician Well-being Index and the 2-item Maslach Burnout Inventory. The Patient Health Questionnaire-2 and Generalized Anxiety Disorder-2 were used to screen for depressive disorders and anxiety disorders, respectively. Associations with sociodemographic and professional characteristics were assessed., Results: Of the 613 surveys administered to trainees and attending physicians, 340 were completed (56%). Among 154 trainees, distress was present in 49%, professional burnout in 35%, positive depressive disorder screening in 5%, and positive anxiety disorder screening in 16%. In univariable analysis, female gender, hours worked in a typical week (HW), and nights on call in a typical week (NOC) were significantly associated with distress. In multivariable analysis, female gender (odds ratio, 3.91; P = .001) and HW (odds ratio for each 10 HW, 1.89; P = .003) remained significantly associated with distress. Female gender, HW, and NOC were significantly associated with burnout univariably, although only HW (odds ratio for each 10 HW, 1.92; P = .003) remained significantly associated with burnout in a multivariable setting. Attending physicians had less distress than trainees ( P = .02) and felt less callous and less emotionally hardened than trainees ( P < .001)., Conclusion: Otolaryngology trainees experience significant work-place distress (49%) and burnout (35%). Gender, HW, and NOC had the strongest associations with distress and burnout.

Objective: To ascertain the prevalence of and associations with distress and professional burnout among academic otolaryngology attending physicians., Study Design: Cross-sectional survey., Setting: Twelve US academic otolaryngology programs., Methods: A questionnaire was administered that encompassed sociodemographic and professional features, the Expanded Physician Well-being Index for distress, the 2-item Maslach Burnout Inventory for professional burnout, the Patient Health Questionnaire-2 screen for major depressive disorder, and the Generalized Anxiety Disorder-2 screen for generalized anxiety disorder., Results: The survey response rate was 56% and included 186 attending physicians. The average respondent age was 47 years; 72% were men; 93% were married or partnered; and 86% had children. Distress was present in 40%, professional burnout in 26%, positive depression screening in 8%, and positive anxiety screening in 11%. In a univariable setting, age, hours worked in a typical week, nights on call in a typical week, and years of practice were significantly associated with distress, although in a multivariable setting, only hours worked in a typical week remained significantly associated with a positive Expanded Physician Well-being Index screen (odds ratio for each 10-hour increase, 2.61; 95% CI, 1.73-3.93; P < .001). In a univariable setting, hours worked in a typical week was significantly associated with a positive Maslach Burnout Inventory screen., Conclusion: Distress or professional burnout occurs in more than a quarter of academic otolaryngology attending physicians, whereas the prevalence of depression or anxiety is approximately 10%. The number of hours worked per week had the strongest association with distress and burnout. These findings may be used to develop and implement programs to promote physician well-being and mitigate professional burnout.

Context.—: One goal of the joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee is to ensure the accurate detection and description of chromosomal abnormalities in both constitutional and neoplastic specimens, including hematologic neoplasms., Objective.—: To report a 20-year performance summary (1999-2018) of conventional chromosome challenges focusing on hematologic neoplasms., Design.—: A retrospective review was performed from 1999 through 2018 to identify karyotype challenges specifically addressing hematologic neoplasms. The overall performance of participants was examined to identify potential recurring errors of clinical significance., Results.—: Of 288 total conventional chromosome challenges from 1999-2018, 87 (30.2%) were presented in the context of a hematologic neoplasm, based on the provided clinical history, specimen type, and/or chromosomal abnormalities. For these 87 hematologic neoplasm challenges, 91 individual cases were provided and graded on the basis of abnormality recognition and karyotype nomenclature (ISCN, International System for Human Cytogenomic [previously Cytogenetic] Nomenclature). Of the 91 cases, 89 (97.8%) and 87 (95.6%) exceeded the required 80% consensus for grading of abnormality recognition and correct karyotype nomenclature, respectively. The 2 cases (2 of 91; 2.2%) that failed to meet the 80% consensus for abnormality recognition had complex karyotypes. The 4 cases (4 of 91; 4.4%) that failed to meet the 80% consensus for correct karyotype nomenclature were the result of incorrect abnormality recognition (2 cases), missing brackets in the karyotype (1 case), and incorrect breakpoint designation (1 case)., Conclusions.—: This 20-year review demonstrates clinical cytogenetics laboratories have been and continue to be highly proficient in the detection and description of chromosomal abnormalities associated with hematologic neoplasms., Competing Interests: Yuhas and Kearney coordinate the vendor series to the College of American Pathologists and receive royalties for this work. The other authors have no relevant financial interest in the products or companies described in this article.

Background: Trimethylamine- N -oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. We sought to determine: (1) TMAO response to meals containing free versus lipid-soluble choline and (2) effects of gut microbiome on TMAO response., Methods: In a randomized, controlled, double-blinded, crossover study, healthy men ( n = 37) were provided meals containing 600 mg choline either as choline bitartrate or phosphatidylcholine, or no choline control., Results: Choline bitartrate yielded three-times greater plasma TMAO AUC ( p = 0.01) and 2.5-times greater urinary TMAO change from baseline ( p = 0.01) compared to no choline and phosphatidylcholine. Gut microbiota composition differed (permutational multivariate analysis of variance, PERMANOVA; p = 0.01) between high-TMAO producers (with ≥40% increase in urinary TMAO response to choline bitartrate) and low-TMAO producers (with <40% increase in TMAO response). High-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae compared to low-TMAO producers (analysis of composition of microbiomes, ANCOM; p < 0.05)., Conclusion: Given that phosphatidylcholine is the major form of choline in food, the absence of TMAO elevation with phosphatidylcholine counters arguments that phosphatidylcholine should be avoided due to TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiome may be effective in reducing disease risk.

This study evaluates newly diagnosed IgM (6%, n = 75/1174) vs. non-IgM light chain amyloidosis patients. IgM amyloid patients had lower light chains (12.5 vs. 22.5 mg/dL; p < 0.001). Heart (56% vs. 73%, p = 0.002) and >1 organ involvement (31% vs. 44%, p = 0.02) was less common in IgM amyloidosis, while soft tissue and peripheral nerve involvement was more common. t(11;14) was less common (27% vs. 50%, p = 0.008) in IgM amyloidosis. Rates of MYD88 L265P and CXCR4 WHIM mutation in IgM amyloidosis were 58% (29/50) and 17% (8/46). Diagnosis after hematopathology review in IgM amyloidosis was pure plasma cell neoplasm (PPCN) in 23% (16/70), lymphoplasmacytic neoplasm (LPL) in 63% (44/70) patients, and other (14%). LPL vs. PPCN groups had distinct genetic abnormalities: t(11;14): 0% (0/18) vs. 60% (9/15), p < 0.001; MYD88 L265P mutation: 84% (27/32) vs. 0% (0/14), p < 0.001; CXCR4 mutation: 29% (8/28) vs. 0% (0/14), p = 0.04. Overall survival was shorter in IgM AL when stratified by Mayo 2012 stage; stage 1/2 (59 vs. 125.9 months, p = 0.003) and stage 3/4 (6.5 vs. 12.9 months, p = 0.075), likely due to lower hematologic response rates (6 months: 39% vs. 59%, p = 0.008). We characterized two subtypes of IgM amyloidosis (LPL/PPCN). This can aid in therapeutic decision-making, with treatment directed at the clonal disease.

Importance: Idiopathic subglottic stenosis (iSGS) is a progressive and potentially life-threatening condition with very few targeted treatment options., Objective: To characterize the clinical factors of patients with iSGS, including body mass index (BMI, calculated as weight in kilograms divided by height in meters squared), and evaluate their association with iSGS symptomatic recurrence., Design, Setting, and Participants: This retrospective medical record review included 186 adult patients with iSGS treated at a single tertiary referral center between January 1, 1989, and December 31, 2015. All data analysis took place from January 1, 2018 to June 30, 2018., Main Outcomes and Measures: The 3 BMI categories were examined for their association with iSGS recurrence. Outcome measurements included time to first symptomatic recurrence (TTFR) and recurrence-free survival (RFS). Comorbidities were recorded., Results: Of the 186 patients in the study, 182 (98%) were women; mean (interquartile range) patient age, 49 (41-60) years. At iSGS diagnosis, 65 (35%) patients were underweight or normal weight; 45 (24%) were overweight; and 76 (41%) were obese (class 1, 2, or 3). Median BMI was 27.4. Ninety-one patients experienced TTFR at a median of 14 months. Compared with underweight or normal-weight patients, the hazard ratios for the associations of overweight, obese class 1, and obese class 2/3 patients with recurrence were 1.14 (95% CI, 0.65-1.99), 1.74 (95% CI, 1.04-2.93), and 1.04 (95% CI, 0.54-1.99), respectively. No differences in concomitant medical treatment regimens were found. While several comorbidities (gastroesophageal reflux disease, hypertension, hyperlipidemia, and diabetes mellitus) were associated with increasing BMI, they were not associated with iSGS symptomatic recurrence on multivariable analysis., Conclusions and Relevance: Results of this retrospective review show that class 1 obesity was associated with an increased rate of iSGS symptomatic recurrence compared with underweight or normal-weight patients. This association was not seen in class 2 or class 3 obesity. Patients with class 1 obesity should be counseled about this risk to aid in the assessment and management of symptoms.

Objectives: This study aimed to characterise the presenting features and outcomes of patients with vasculitis and gastrointestinal perforation., Methods: Using a retrospective cohort design, this study included 20 cases with verified vasculitis and gastrointestinal perforation at Mayo Clinic, Rochester, USA, between 1998 and 2017., Results: Four of the twenty cases experienced vasculitis-induced perforation. Cases with perforations due to vasculitic involvement had more small bowel involvement, longer duration of abdominal pain prior to perforation (41 days vs. 0 days, p=0.005), and a higher proportion of active tobacco use (75% vs. 7%, p=0.01) compared to the cases with non-vasculitis perforation. A majority (88%) of the non-vasculitis perforations were associated with glucocorticoid use. The median cumulative glucocorticoid dose prior to perforation in patients with additional, non-vasculitic risk factors for perforation was 4,320 mg prednisone and was 22,170 mg for those without additional risk factors. Mortality rates for the whole cohort were higher than the general population (standardised mortality ratio: 2.19, 95% confidence interval 1.05 to 4.02). The cases with vasculitis-induced perforation tended to have increased number of surgeries and length of stay compared to the non-vasculitis cases; however, those differences failed to reach statistical significance., Conclusions: Small bowel location and longer abdominal pain duration may help distinguish vasculitis-induced bowel perforation from other etiologies. Overall mortality in patients with vasculitis and bowel perforation is increased, highlighting the importance of prompt diagnosis and management.

In this study, we determined the impact of the total Western diet (TWD) for rodents and its macro- and micronutrient components on weight gain and biomarkers of metabolic function in mice compared to a 45% fat diet-induced obesity (DIO) diet and the standard AIN93G diet. We hypothesized that mice fed the TWD would have increased body fat with indicators of metabolic syndrome similar to mice consuming the DIO diet. As expected, DIO-fed mice acquired a metabolic syndrome phenotype typified by increased energy intake, increased body weight gain, increased fat mass, higher fasting glucose, impaired glucose tolerance, and higher plasma leptin relative to the AIN93G diet. Mice fed a macronutrient-modified (MM) diet (with standard vitamin and mineral composition) had a similar response, albeit to a lesser degree than mice fed the DIO diet. Mice fed a vitamin- and mineral-modified diet (with standard macronutrient composition) were not different from mice fed the AIN93G diet. Surprisingly, the TWD (with modified macronutrients, vitamins and minerals) did not significantly affect any of these parameters, despite the fact that the TWD macronutrient profile was identical to the MM diet. These data suggest that, in the context of the TWD, vitamin and mineral intakes in mice that reflect a Western dietary pattern inhibit the hyperphagia and resulting increased weight gain associated with the higher fat content of the TWD. In conclusion, these observations underscore the need to consider the influence of micronutrient intakes in pre-clinical models of obesity and metabolic syndrome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The purpose of this study was to describe and evaluate a newly developed on-ice timing system for team evaluation in the sport of ice hockey. We hypothesized that this new, simple, inexpensive, timing system would prove to be highly accurate and reliable. Six adult subjects (age 30.4 ± 6.2 years) performed on ice tests of acceleration and conditioning. The performance times of the subjects were recorded using a handheld stopwatch, photocell, and high-speed (240 frames per second) video. These results were then compared to allow for accuracy calculations of the stopwatch and video as compared with filtered photocell timing that was used as the "gold standard." Accuracy was evaluated using maximal differences, typical error/coefficient of variation (CV), and intraclass correlation coefficients (ICCs) between the timing methods. The reliability of the video method was evaluated using the same variables in a test-retest analysis both within and between evaluators. The video timing method proved to be both highly accurate (ICC: 0.96-0.99 and CV: 0.1-0.6% as compared with the photocell method) and reliable (ICC and CV within and between evaluators: 0.99 and 0.08%, respectively). This video-based timing method provides a very rapid means of collecting a high volume of very accurate and reliable on-ice measures of skating speed and conditioning, and can easily be adapted to other testing surfaces and parameters.

Background: Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV infection, but the pathogenic mechanisms contributing to the lethal disease are unclear., Methods: To gain insights into the pathogenesis of TCRV infection in AG129 mice, we assessed hematologic and cytokine responses during the course of infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute infection, and measured serum and tissue viral titers out to 40 days post-infection to determine whether the virus is ultimately cleared in recovering mice., Results: We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2-3 weeks post-infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study., Conclusions: Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 microg/mL.

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.

A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

The preparation and characterization of a series of C-10 substituted 5-allyl-2,5-dihydro-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines as a novel class of selective ligands for the glucocorticoid receptor is described. Substitution at the C-10 position of the tetracyclic core with linear, two-atom appendages (OCH(3), OCF(2)H, NHMe, SMe, CH=CH(2), Ctbd1;CH, CH(2)OH) provided molecules of high affinity (K(i) = 2-8 nM) for the human glucocorticoid receptor (hGR) with limited cross-reactivity with other steroid receptors (PR, MR, AR, ER). Optimal analogues showed slightly less potent but highly efficacious E-selectin repression with reduced levels of GRE activation efficacy in reporter gene assays relative to prednisolone. Preliminary SAR of analogues containing substitution at the C-9 and C-10 positions identified the 9-OH, 10-OMe analogue 50 and the 9-OH, 10-Cl analogue 58 as compounds that demonstrated potent, GR-mediated inhibition in a conconavalin A stimulated T-cell proliferation assay in both rodent and human whole blood monocytes. When evaluated for their in vivo effects in carrageenan-induced paw edema in rats, 50, 58, and 10-OCF(2)H analogue 35 showed dose-dependent anti-inflammatory effects (50, ED(50) = 16 mg/kg; 58, ED(50) = 15 mg/kg; 35, ED(50) = 21 mg/kg vs ED(50) = 15 mg/kg for 18 and ED(50) = 4 mg/kg for prednisolone).

Tricolorin A (1) is a novel tetrasaccharide macrolactone that is a natural herbicide. In this paper is reported a total synthesis of 1. Coupling of hydroxy ester 18 with D-fucosyl trichloroacetimidate 23 gave fucoside 24. Removal of the C-2 pivaloyl group of 24 followed by coupling with D-glucosyl trichloroacetimidate 29 resulted in isolation of disaccharide 30. Saponification of the ester groups of 30 and subsequent selective macrolactonization of the acid diol 31 by the Yonemitsu protocol gave only the desired lactone 32. The key step in the assembly of disaccharide glycosyl trichloroacetimidate 52 was coupling L-rhamnoside 47 with L-rhamnosyl trichloroacetimidate 43. Attempts to couple lactone disaccharide 32 with disaccharide 52 were unsuccessful. Using an alternate plan for assembly of the tetrasaccharide, reaction of disaccharide glycosyl trichloroacetimidate 58 with disaccharide 37 gave tetrasaccharide 59. Diester lactone 63 was generated by selective macrolactonization of tetrasaccharide acid triol 60, again using the Yonemitsu protocol, followed by addition of the chiral side chain acid to the reaction vessel. Synthetic tricolorin A (1) was obtained by deprotection of 63. Starting from fucose, glucose, rhamnose, and (S)-1-octyn-3-ol, the synthesis required 39 steps overall. The longest linear sequence was 14 steps, with an overall yield for this longest linear sequence of 6%.