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Melinda M Somasekhar1, Alfred Bove2, Chris Rausch1, James Degnan3, Cathy T King1, Arnold Meyer11The Albert J Finestone, MD, Office for Continuing Medical Education, 2Section of Cardiology, Temple University School of Medicine, Philadelphia, PA, USA; 3Measurement and Research Center, Temple University, Philadelphia, PA, USAAbstract: Cost related to higher-level outcomes measurement is often very high. However, the cost burden is felt even more by smaller, less well-funded continuing medical education (CME) programs. It is possible to overcome financial and participant-related barriers to measuring Level 6 outcomes, which are patient health outcomes. The Temple University School of Medicine’s Office for Continuing Medical Education developed a sequential tool for attaining cost-effective outcomes measurement for determining the likelihood of a CME intervention to produce significant changes in physician performance. The appropriate selection of the CME topic and specific practice change indictors drive this tool. This tool walks providers through a simple YES or NO decision-making list that guides them toward an accurate prediction of potential programmatic outcomes. Factors considered during the decision-making process include whether: (a) the intended change(s) will have a substantial impact on current practice; (b) the intended practice change(s) are well supported by clinical data, specialty organization/government recommendations, expert opinion, etc; (c) the potential change(s) affects a large population; (d) external factors, such as system pressures, media pressures, financial pressures, patient pressures, safety pressures, etc, are driving this intended change in performance; (e) there is a strong motivation on the part of physicians to implement the intended change(s); and (f) the intended change(s) is relatively easy to implement within any system of practice. If each of these questions can be responded to positively, there is a higher likelihood that the intended practice-related change(s) will occur. Such change can be measured using a simpler and less costly methodology.Keywords: outcomes, outcomes measurement, cost-effective, evaluation tool, continuing medical education

Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4 + and CD8 + T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4 + T-cell subsets. Immune activation was significantly higher in all CD8 + T-cell subsets, and memory CD8 + T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8 + T-cells. The differences seen in CM and TM CD4 + T-cell subsets were more pronounced among participants with CD4 + T-cell counts of <500 cells/μL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4 + T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4 + and CD8 + T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4 + T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4 + and CD8 + T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.

Within vascular plants, the partitioning of hydraulic resistance along the soil-to-leaf continuum affects transpiration and its response to environmental conditions. In trees, the fractional contribution of leaf hydraulic resistance (R leaf ) to total soil-to-leaf hydraulic resistance (R total ), or fR leaf (=R leaf /R total ), is thought to be large, but this has not been tested comprehensively. We compiled a multibiome data set of fR leaf using new and previously published measurements of pressure differences within trees in situ. Across 80 samples, fR leaf averaged 0.51 (95% confidence interval [CI] = 0.46-0.57) and it declined with tree height. We also used the allometric relationship between field-based measurements of soil-to-leaf hydraulic conductance and laboratory-based measurements of leaf hydraulic conductance to compute the average fR leaf for 19 tree samples, which was 0.40 (95% CI = 0.29-0.56). The in situ technique produces a more accurate descriptor of fR leaf because it accounts for dynamic leaf hydraulic conductance. Both approaches demonstrate the outsized role of leaves in controlling tree hydrodynamics. A larger fR leaf may help stems from loss of hydraulic conductance. Thus, the decline in fR leaf with tree height would contribute to greater drought vulnerability in taller trees and potentially to their observed disproportionate drought mortality., (© 2022 John Wiley & Sons Ltd.)

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α + ) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α + breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α + BC cell lines. MCF-7 ER-α + BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α + BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α + secretome. In contrast to ER-α + CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α + BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues., (© 2022. The Author(s).)

Postingestive signals are important for shaping appetitive and consummatory responses, but the brain mechanisms required to assimilate interoceptive events with those at the frontlines of ingestion (taste-guided) are poorly understood. Here, we investigated whether an insular cortex (IC) region, which receives viscerosensory input, including gustatory, is required to modify taste-elicited consummatory reactions in response to a real-time interoceptive change using a serial taste reactivity (TR) test where the rats' oromotor and somatic reactions to intraoral (IO) infusions of sucrose were periodically assessed over 45 min following lithium chloride (LiCl) administration. Results showed that neurally-intact rats shifted from an ingestive repertoire to an aversive one as LiCl took effect. Overall, this hedonic shift was delayed in rats with bilateral neurotoxic IC lesions. Rats with greater neuronal loss in posterior gustatory IC displayed fewer aversive reactions to sucrose following this initial LiCl injection. We further assessed whether the failure to integrate interoceptive feedback with ongoing taste-guided behavior impaired acquisition and/or expression of conditioned aversion and/or avoidance in these same rats. Although, as a group, LiCl-injected rats with IC lesions subsequently avoided the sugar in a 48-h two-bottle test, their preference for sucrose was significantly greater than that of the LiCl-injected neurally-intact rats. Overall lesion size, as well as proportion of the posterior gustatory and/or anterior visceral IC were each associated with impaired avoidance. These findings reveal new roles for the posterior gustatory and anterior visceral ICs in multisensory integrative function., (Copyright © 2022 Jung et al.)

Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1's role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 King, Matossian, Savoie, Nguyen, Wright, Byrne, Elliott, Burks, Bratton, Pashos, Bunnell, Burow, Collins-Burow and Martin.)

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell-matrix interactions in rare cancer types using higher passage PDX tissue., (© 2021. The Author(s).)

Clinical outcomes are inferior for individuals with HIV having suboptimal CD4 T-cell recovery during antiretroviral therapy (ART). We investigated if the levels of infection and the response to homeostatic cytokines of CD4 T-cell subsets contributed to divergent CD4 T-cell recovery and HIV reservoir during ART by studying virologically-suppressed immunologic responders (IR, achieving a CD4 cell count >500 cells/μL on or before two years after ART initiation), and virologically-suppressed suboptimal responders (ISR, did not achieve a CD4 cell count >500 cells/μL in the first two years after ART initiation). Compared to IR, ISR demonstrated higher levels of HIV-DNA in naïve, central (CM), transitional (TM), and effector (EM) memory CD4 T-cells in blood, both pre- and on-ART, and specifically in CM CD4 T-cells in LN on-ART. Furthermore, ISR had higher pre-ART plasma levels of IL-7 and IL-15, cytokines regulating T-cell homeostasis. Notably, pre-ART PD-1 and TIGIT expression levels were higher in blood CM and TM CD4 T-cells for ISR; this was associated with a significantly lower fold-changes in HIV-DNA levels between pre- and on-ART time points exclusively on CM and TM T-cell subsets, but not naïve or EM T-cells. Finally, the frequency of CM CD4 T-cells expressing PD-1 or TIGIT pre-ART as well as plasma levels of IL-7 and IL-15 predicted HIV-DNA content on-ART. Our results establish the association between infection, T-cell homeostasis, and expression of PD-1 and TIGIT in long-lived CD4 T-cell subsets prior to ART with CD4 T-cell recovery and HIV persistence on-ART., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: V.C.M. has received investigator initiated research grants (to the institution, unrelated to current work) from Bayer, Gilead Sciences, Lilly and ViiV. All other authors have declared that no competing interests exist.

The development of resistance to therapy is a significant obstacle to effective therapeutic regimens. Evaluating the effects of oncology drugs in the laboratory setting is limited by the lack of translational models that accurately recapitulate cell-microenvironment interactions present in tumors. Acquisition of resistance to therapy is facilitated, in part, by the composition of the tumor extracellular matrix (ECM), with the primary current in vitro model using collagen I (COL I). Here we seek to identify the prevalence of COL I-enhanced expression in the triple-negative breast cancer (TNBC) subtype. Furthermore, we identify if methods of response to therapy are altered depending on matrix composition. We demonstrated that collagen content varies in patient tumor samples across subtypes, with COL I expression dramatically increased in typically less aggressive estrogen receptor (ER)-positive(ER + )/progesterone receptor (PGR)-positive (PGR + ) cancers irrespective of patient age or race. These findings are of significance considering how frequently COL I is implicated in tumor progression. In vitro analyses of ER + and ER-negative (ER - ) cell lines were used to determine the effects of ECM content (collagen I, collagen IV, fibronectin, and laminin) on proliferation, cellular phenotype, and survival. Neither ER + nor ER - cells demonstrated significant increases in proliferation when cultured on these ECM substrates. ER - cells cultured on these substrates were sensitized to both chemotherapy and targeted therapy. In addition, MDA-MB-231 cells expressed different morphologies, binding affinities, and stiffness across these substrates. We also demonstrated that ECM composition significantly alters transcription of senescence-associated pathways across ER + and ER - cell lines. Together, these results suggest that complex matrix composites should be incorporated into in vitro tumor models, especially for the drug-resistant TNBC subtype. Impact statement The importance of tumor extracellular matrix (ECM) in disease progression is often inadequately represented in models of breast cancer that rely heavily on collagen I and Matrigel. Through immunohistochemistry analysis of patient breast tumors, we show a wide variation in collagen content based on subtype, specifically a repression of fibril collagens in the receptor negative subtype, irrespective of age and race. We also demonstrated that tumor ECM composition alters cellular elasticity and oncogenic pathway activation demonstrating that physiologically relevant three-dimensional models of breast cancer should include an ECM that is subtype specific.

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer., (Copyright © 2020 Hoang, Matossian, Ucar, Elliott, La, Wright, Burks, Perles, Hossain, King, Browning, Bursavich, Fang, Del Valle, Bhatt, Cavanaugh, Flaherty, Anbalagan, Rowan, Bratton, Nephew, Miele, Collins-Burow, Martin and Burow.)

Lymph nodes (LN) and their resident T follicular helper CD4+ T cells (Tfh) are a critical site for HIV replication and persistence. Therefore, optimizing antiviral activity in lymphoid tissues will be needed to reduce or eliminate the HIV reservoir. In this study, we retained effector immune cells in LN of cART-suppressed, SIV-infected rhesus macaques by treatment with the lysophospholipid sphingosine-1 phosphate receptor modulator FTY720 (fingolimod). FTY720 was remarkably effective in reducing circulating CD4+ and CD8+ T cells, including those with cytolytic potential, and in increasing the number of these T cells retained in LN, as determined directly in situ by histocytometry and immunohistochemistry. The FTY720-induced inhibition of T cell egress from LN resulted in a measurable decrease of SIV-DNA content in blood as well as in LN Tfh cells in most treated animals. In conclusion, FTY720 administration has the potential to limit viral persistence, including in the critical Tfh cellular reservoir. These findings provide rationale for strategies designed to retain antiviral T cells in lymphoid tissues to target HIV remission., Competing Interests: The authors have declared that no competing interests exist.

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

The bone marrow (BM) is the key anatomic site for hematopoiesis and plays a significant role in the homeostasis of mature T cells. However, very little is known on the phenotype of BM-derived CD4 + T cells, their fate during simian immunodeficiency virus (SIV) infection, and their contribution to viral persistence during antiretroviral therapy (ART). In this study, we characterized the immunologic and virologic status of BM-derived CD4 + T cells in rhesus macaques prior to SIV infection, during the early chronic phase of infection, and during ART. We found that BM memory CD4 + T cells are significantly depleted following SIV infection, at levels that are similar to those measured in the peripheral blood (PB). In addition, BM-derived memory CD4 + T cells include a high frequency of cells that express the coinhibitory receptors CTLA-4 and PD-1, two subsets previously shown to be enriched in the viral reservoir; these cells express Ki-67 at levels similar to or higher than the same cells in PB. Finally, when we analyzed SIV-infected RMs in which viral replication was effectively suppressed by 12 months of ART, we found that BM CD4 + T cells harbor SIV DNA and SIV RNA at levels comparable to those of PB CD4 + T cells, including replication-competent SIV. Thus, BM is a largely understudied anatomic site of the latent reservoir which contributes to viral persistence during ART and needs to be further characterized and targeted when designing therapies for a functional or sterilizing cure to HIV. IMPORTANCE The latent viral reservoir is one of the major obstacles in purging the immune system of HIV. It is paramount that we elucidate which anatomic compartments harbor replication-competent virus, which upon ART interruption results in viral rebound and pathogenesis. In this study, using the rhesus macaque model of SIV infection and ART, we examined the immunologic status of the BM and its role as a potential sanctuary for latent virus. We found that the BM compartment undergoes a similar depletion of memory CD4 + T cells as PB, and during ART treatment the BM-derived memory CD4 + T cells contain high levels of cells expressing CTLA-4 and PD-1, as well as amounts of cell-associated SIV DNA, SIV RNA, and replication-competent virus comparable to those in PB. These results enrich our understanding of which anatomic compartments harbor replication virus and suggest that BM-derived CD4 + T cells need to be targeted by therapeutic strategies aimed at achieving an HIV cure., (Copyright © 2018 American Society for Microbiology.)

A key step in the therapeutic antibody drug discovery process is early identification of diverse candidate molecules. Information comparing antibody binding epitopes can be used to classify antibodies within a large panel, guiding rational lead molecule selection. We describe a novel epitope binning method utilizing high-throughput flow cytometry (HTFC) that leverages cellular barcoding or spectrally distinct beads to multiplex samples to characterize antibodies raised against cell membrane receptor or soluble protein targets. With no requirement for sample purification or direct labeling, the method is suited for early characterization of antibody candidates. This method generates competitive binding profiles of each antibody against a defined set of known or unknown reference antibodies for binding to epitopes of an antigen. Antibodies with closely related competitive binding profiles indicate similar epitopes and are classified in the same bin. These large, high-throughput, multiplexed experiments can yield epitope bins or clusters for the entire antibody panel, from which a conceptual map of the epitope space for each antibody can be created. Combining this valuable epitope information with other data, such as functional activity, sequence, and selectivity of binding to orthologs and paralogs, enables us to advance the best epitope-diverse candidates for further development.

The extracellular matrix (ECM) is a critical cue to direct tumorigenesis and metastasis. Although two-dimensional (2D) culture models have been widely employed to understand breast cancer microenvironments over the past several decades, the 2D models still exhibit limited success. Overwhelming evidence supports that three dimensional (3D), physiologically relevant culture models are required to better understand cancer progression and develop more effective treatment. Such platforms should include cancer-specific architectures, relevant physicochemical signals, stromal-cancer cell interactions, immune components, vascular components, and cell-ECM interactions found in patient tumors. This review briefly summarizes how cancer microenvironments (stromal component, cell-ECM interactions, and molecular modulators) are defined and what emerging technologies (perfusable scaffold, tumor stiffness, supporting cells within tumors and complex patterning) can be utilized to better mimic native-like breast cancer microenvironments. Furthermore, this review emphasizes biophysical properties that differ between primary tumor ECM and tissue sites of metastatic lesions with a focus on matrix modulation of cancer stem cells, providing a rationale for investigation of underexplored ECM proteins that could alter patient prognosis. To engineer breast cancer microenvironments, we categorized technologies into two groups: (1) biochemical factors modulating breast cancer cell-ECM interactions and (2) 3D bioprinting methods and its applications to model breast cancer microenvironments. Biochemical factors include matrix-associated proteins, soluble factors, ECMs, and synthetic biomaterials. For the application of 3D bioprinting, we discuss the transition of 2D patterning to 3D scaffolding with various bioprinting technologies to implement biophysical cues to model breast cancer microenvironments.

The persistence of HIV reservoirs, including latently infected, resting CD4 + T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 + T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 + T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV + or SIV + subjects, we found that most of the circulating memory CD32 + CD4 + T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a T H 2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4 + T cells in peripheral blood or lymphoid tissue; isolated CD32 + resting CD4 + T cells accounted for less than 3% of the total HIV DNA in CD4 + T cells. Cell-associated HIV DNA and RNA loads in CD4 + T cells positively correlated with the frequency of CD32 + CD69 + CD4 + T cells but not with CD32 expression on resting CD4 + T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4 + T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4 + T cells enriched for transcriptionally active HIV after long-term ART., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V 1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V 1.7 DII voltage sensor domain and block human Na V 1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V 1.7 DII voltage sensor domain via ELISA, and does not bind Na V 1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V 1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V 1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V 1.7 target sequences or specifically inhibit Na V 1.7 current., Competing Interests: All authors were full-time employees at Amgen, Inc. at the time the experiments were conducted.

Because of the large number of total knee replacement (TKR) surgeries conducted per year, and with projections of increased demand to almost a million primary TKR surgeries per year by 2030 in the United States alone, there is a need to discover more efficient working materials as alternatives to current bone cements. There is a need for surgeons and hospitals to become more efficient and better control over the operative environment. One area of inefficiency is the cement steps during TKR. Currently the surgeon has very little control over cement polymerization. This leads to an increase in time, waste, and procedural inefficiencies. There is a clear need to create an extended working time, moldable, osteoconductive, and osteoinductive bone augment as a substitution for the current clinically used bone cement where the surgeon has better control over the polymerization process. This study explored several compositions of pentaerythritol-co-trimethylolpropane tris-(3-mercaptopropionate) hydroxyapatite composite materials prepared via benzoyl peroxide-initiated thermal frontal polymerization. The 4:1 acrylate to thiol ratio containing augment material shows promise with a maximal propagation temperature of 160°C ± 10°C, with mechanical strength of 3.65 MPa, and 111% cytocompatibility, relative to the positive control. This frontally polymerized material may have application as an augment with controlled polymerization supporting cemented implants. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1152-1160, 2016., (© 2015 Wiley Periodicals, Inc.)

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Rats display stereotypical oromotor and somatic responses to small volumes of intraorally infused taste solutions. These behaviors, known as taste reactivity, are categorized by their association with ingestion or rejection and are thought to reflect the palatability of the stimulus. Because supracollicular decerebrate rats display normal taste reactivity responses, it would appear that forebrain structures are not necessary for generating them. However, because moving the plane of transection rostrally, or damaging or manipulating specific ventral forebrain sites disrupts normal taste reactivity behavior, lesions of the gustatory cortex, a region that has been suggested to be involved with palatability processing, may do the same. In the current study, rats received two injections of either ibotenic acid (N=12) or vehicle (N=8), targeting the conventionally defined gustatory cortex in each hemisphere, and were implanted with intraoral cannulae. Following recovery, their responses to intraoral infusions (0.23ml in 1min) of dH2O, sucrose (1.0M and 0.1M), and quinine hydrochloride (3mM and 0.3mM) were video recorded. Analysis of brains with sufficient bilateral lesions (N=10) revealed that, on average, approximately 94% of the gustatory cortex was destroyed. These extensive bilateral lesions had no significant effect on taste reactivity; the numbers of ingestive and aversive responses to sucrose and quinine were similar between groups. Though these findings do not rule out involvement of the gustatory cortex in palatability processing, they make evident that the region of insular cortex destroyed is not necessary for the normal expression of unconditioned affective behavioral responses to taste stimuli., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Remarkably, when lingual gustatory nerves are surgically rerouted to inappropriate taste fields in the tongue, some taste functions recover. We previously demonstrated that quinine-stimulated oromotor rejection reflexes and neural activity (assessed by Fos immunoreactivity) in subregions of hindbrain gustatory nuclei were restored if the posterior tongue, which contains receptor cells that respond strongly to bitter compounds, was cross-reinnervated by the chorda tympani nerve. Such functional recovery was not seen if instead, the anterior tongue, where receptor cells are less responsive to bitter compounds, was cross-reinnervated by the glossopharyngeal nerve, even though this nerve typically responds robustly to bitter substances. Thus, recovery depended more on the taste field being reinnervated than on the nerve itself. Here, the distribution of quinine-stimulated Fos-immunoreactive neurons in two taste-associated forebrain areas was examined in these same rats. In the central nucleus of the amygdala (CeA), a rostrocaudal gradient characterized the normal quinine-stimulated Fos response, with the greatest number of labeled cells situated rostrally. Quinine-stimulated neurons were found throughout the gustatory cortex, but a "hot spot" was observed in its anterior-posterior center in subregions approximating the dysgranular/agranular layers. Fos neurons here and in the rostral CeA were highly correlated with quinine-elicited gapes. Denervation of the posterior tongue eliminated, and its reinnervation by either nerve restored, numbers of quinine-stimulated labeled cells in the rostralmost CeA and in the subregion approximating the dysgranular gustatory cortex. These results underscore the remarkable plasticity of the gustatory system and also help clarify the functional anatomy of neural circuits activated by bitter taste stimulation., (© 2014 Wiley Periodicals, Inc.)

Objective: Due to the rolling manufacturing process, most advanced high-strength steels (AHSS) demonstrate in-plane anisotropic material behavior. This study investigates the effects of material orientation on the axial crush behavior and fracture of AHSS with axial crush tests and computer simulations., Methods: Crush simulation models considering material anisotropy and damage evolution were developed in LS-DYNA based on the drop-tower crush test results and coupon characterization test data for DP780 steel. The modified Mohr-Coulomb (MMC) isotropic fracture model was employed in the crush simulation models for fracture prediction., Results: The 12-sided components fabricated in the transverse (T) direction of the sheet exhibited slightly higher crush loads and reduced crush distances compared to those in the longitudinal (L) direction. The crush behavior in each direction was generally proportional to ultimate tensile strength. All of the materials investigated in this study showed some cracking in the crush tests for both component orientations, but only DP780 showed significant anisotropy in fracture behavior with more cracking for the T direction compared to the L direction. Overall, the amount of cracking observed in the tests had little or no significant effect on the axial crush performance. The MMC fracture loci in both the L and T directions were determined using a reverse engineering approach, and the stress-strain curves beyond the uniform elongation point were extended using an optimization method. Both material models MAT103 and MAT224 predicted the crush and fracture behavior with reasonably good accuracy., Conclusions: The predicted fracture mode and force-displacement curves agreed well with the test data for both the L and T directions in axial crush tests of the 12-sided components. The simple isotropic material model MAT224 is adequate for crush simulations to predict material orientation effects on AHSS component crush performance and fracture behavior.

Studies examining the effects of transection and regeneration of the glossopharyngeal (GL) and chorda tympani (CT) nerves on various taste-elicited behaviors in rats have demonstrated that the GL (but not the CT) nerve is essential for the maintenance of both an unconditioned protective reflex (gaping) and the neural activity observed in central gustatory structures in response to lingual application of a bitter substance. An unresolved issue, however, is whether recovery depends more on the taste nerve and the central circuits that it supplies and/or on the tongue receptor cell field being innervated. To address this question, we experimentally cross-wired these taste nerves, which, remarkably, can regenerate into parts of the tongue they normally do not innervate. We report that quinine-stimulated gaping behavior was fully restored, and neuronal activity, as assessed by Fos immunohistochemistry in the nucleus of the solitary tract and the parabrachial nucleus, was partially restored only if the posterior tongue (PT) taste receptor cell field was reinnervated; the particular taste nerve supplying the input was inconsequential to the recovery of function. Thus, PT taste receptor cells appear to play a privileged role in triggering unconditioned gaping to bitter tasting stimuli, regardless of which lingual gustatory nerve innervates them. Our findings demonstrate that even when a lingual gustatory nerve (the CT) forms connections with taste cells in a non-native receptor field (the PT), unconditioned taste rejection reflexes to quinine can be maintained. These findings underscore the extraordinary ability of the gustatory system to adapt to peripherally reorganized input for particular behaviors.

Introduction: Heightened concerns about industry influence on continuing medical education (CME) have prompted tighter controls on the management of commercial funding and conflict of interest. As a result, CME providers must closely monitor their activities and intervene if bias or noncompliance with accreditation standards is likely. Potential for industry influence can be difficult to assess at a stage in the planning process when mitigation strategies can assure balance and content validity. Few tools exist to aid providers in this regard., Methods: A 12-item instrument was designed to assess risk for commercial influence on CME. To determine reliability and validity, a cohort of experienced CME professionals applied the tool to standardized "cases" representing CME activities in the early stages of planning. Results were compared with the experts' assignment of the same cases to one of four risk categories. A survey of study participants was conducted to ascertain usefulness and potential applications of the tool., Results: Analysis demonstrated strong intraclass correlation across cases (0.90), interrater reliability (94%), and correlation between assessment of risk with and without the tool (Spearman coefficient, 0.93, p < 0.01; weighted kappa, 0.59). Participants found the tool easy to use and of potential benefit to their CME office., Discussion: The Consortium for Academic Continuing Medical Education (CACME) risk stratification tool can help CME providers identify activities that must be closely monitored for potential industry influence, remain aware of factors that place programming at risk for noncompliance with accreditation standards, and substantiate the allocation of resources by the CME office.

The "waist" area and external subnuclei of the parabrachial nucleus (PBN) have been implicated in the processing of gustatory information, yet their behavioral roles are not clearly defined. In the current study, areas within and surrounding the PBN were stimulated while oromotor behaviors were monitored in conscious rats. Electrical and chemical (100 mM glutamate) stimulation of the waist area increased ingestive oromotor behaviors over baseline (p<.01). Stimulation of external PBN subnuclei and areas medial and ventral to the PBN failed to cause a behavioral change. These data support the hypothesis that the waist area of the PBN constitutes part of the neural substrate involved in eliciting oromotor behaviors in response to taste input.

The distribution of quinine-stimulated Fos-like immunoreactivity (FLI) in several subdivisions of the parabrachial nucleus (PBN) known to be responsive to gustatory stimulation was examined in rats in which the chorda tympani nerve (CT) and/or glossopharyngeal nerve (GL) was transected (Experiment 1) and in rats in which the GL was transected with regeneration promoted or prevented (Experiment 2). We confirmed previous findings in the literature by demonstrating that rats intraorally infused with 3 mM quinine showed a robust population of FLI in the waist area and the external lateral (EL) and external medial (EM) subdivisions of the PBN (Yamamoto et al. [1994] Physiol Behav 56:1197-1202; Travers et al., [ 1999] Am J Physiol 277:R384-R394). In the waist area, only GL transection significantly decreased the number of FLI-neurons elicited by intraoral infusion of quinine compared with water-stimulated controls. In the external subdivisions neither neurotomy affected the number of FLI-neurons. The effect of GL transection in the waist area was enduring for rats in which the GL did not regenerate (up to 94 days), but regeneration of the GL after 52 days restored quinine-stimulated FLI to control values. In these same GL-transected animals, there were parallel decreases in the number of gapes elicited by intraoral quinine stimulation that recovered, but only subsequent to regeneration of the GL. These data provide support for the role of the waist area in the brainstem processing that underlies oromotor rejection behaviors and also help substantiate the hypothesis that the CT and GL are relatively specialized with regard to function. Moreover, when the quinine-induced pattern of neural activity in the second central gustatory relay, as assessed by FLI, is substantially altered by the loss of peripheral gustatory input from the GL, it can be restored upon regeneration of the nerve., (Copyright 2003 Wiley-Liss, Inc.)

Objectives: To (1) describe the rate of glycosylated hemoglobin (HbA1c) testing and control in a primary care clinic at an academic medical center; (2) compare academic medical center and health maintenance organization (HMO) data for a subgroup of the same patients; and (3) discuss the challenges to accurate clinical practice profiling., Study Design: Retrospective analysis of medical records and claims and administrative data., Patients and Methods: We reviewed the medical records of 300 patients with diabetes mellitus from the Primary Care practice at an academic medical center (AMC). The HMO and AMC were in the southeastern United States. The study adhered to the National Committee for Quality Assurance's Health Plan Employer Data and Information Set 3.0 (1998) guidelines for collection of the numerator and denominator data., Results: Overall, 275 (91.7%) of 300 patients underwent a documented HbA1c test (range, 77.8%-98.0% across the 5 Primary Care Associates clinic physicians). Of these 275 patients, 206 (74.9%; range, 52.4%-84.7%) were under "good control" (defined by the National Committee for Quality Assurance as an HbA1c level of < or = 9.5%). The mean (SD) HbA1c level was 8.45% (1.98%). Data from the HMO documented a rate of HbA1c testing of 36.3% (45/124) compared with 92.7% (115/124) based on case note review at the academic medical center., Conclusions: Current administrative and claims-based information systems have inherent weaknesses if used for performance measurement. Reliance solely on medical record review is time and cost prohibitive. To ensure complete reporting of mandated "quality measures" will necessitate the tracking of data across different healthcare systems.

The peripheral, central, and behavioral consequences of glossopharyngeal nerve transection (GLX), regeneration, and the prevention of regeneration on the quinine-elicited responses of adult rats were concurrently examined. Oromotor taste reactivity (TR) was videotaped during intraoral infusion of 7 ml of either quinine (3 mm) or distilled water at 17, 52, or 94 d after surgery. We confirmed previous findings by showing that 17 d after neurotomy, (1) the number of circumvallate (CV) and foliate taste buds, (2) gapes (a characteristic aversive TR response), and (3) the number of Fos-like immunoreactive (FLI) neurons in the gustatory NST (gNST), particularly in the medial portion (subfield 5) of the rostral central subdivision (RC), were all severely attenuated in GLX rats. We extended these findings by showing that these lesion-induced effects were enduring when the GL did not regenerate (up to 94 d). In contrast, when the GL regenerated, as few as 52 d were sufficient to re-establish quinine-elicited TR, especially gaping, and FLI expression in RC, particularly within subfield 5, to values comparable with quinine-stimulated sham-operated rats. Evidently, the gNST maintains its potential to restore accurately the organization of neural activity that is disrupted by nerve injury, as assessed by FLI, ultimately leading to the return of normal protective oromotor responses, provided the nerve regenerates. This recovery was complete despite the reappearance of a reduced population of CV taste buds ( approximately 75% control values) and may relate to peripheral and/or central changes that occur in tandem with regeneration of the GL.

The relationship between specific gustatory nerve activity and central patterns of taste-evoked neuronal activation is poorly understood. To address this issue within the first central synaptic relay in the gustatory system, we examined the distribution of neurons in the nucleus of the solitary tract (NST) activated by the intraoral infusion of quinine using Fos immunohistochemistry in rats with bilateral transection of the chorda tympani (CTX), bilateral transection of the glossopharyngeal nerve (GLX), or combined neurotomy (DBLX). Compared with nonstimulated and water-stimulated controls, quinine evoked significantly more Fos-like-immunoreactive (FLI) neurons across the rostrocaudal extent of the gustatory NST (gNST), especially within its dorsomedial portion (subfield 5). Although the somatosensory aspects of fluid stimulation contributed to the observed increase in FLI neurons, the elevated number and spatial distribution of FLI neurons in response to quinine were remarkably distinguishable from those in response to water. GLX and DBLX produced a dramatic attenuation of quinine-evoked FLI neurons and a shift in their spatial distribution such that their number and pattern were indiscernable from those observed in water-stimulated controls. Although CTX had no effect on the number of quinine-evoked FLI neurons within subfield 5 at intermediate levels of the gNST, it produced intermediate effects elsewhere; yet, the spatial distribution of the quinine-evoked FLI neurons was not altered by CTX. These findings suggest that the GL provides input to all FLI neurons responsive to quinine, however, some degree of convergence with CT input apparently occurs in this subpopulation of neurons. Although the role of these FLI neurons in taste-guided behavioral responses to quinine remains speculative, their possible function in oromotor reflex control is considered.

Careful consideration of the benefit to the mother and the risk to the fetus is required when prescribing antifungal therapy in pregnancy. Imidazoles are considered safe as topical therapy for fungal skin infections during pregnancy. Nystatin is minimally absorbed and is effective for vaginal therapy. Although vaginal use of the imidazoles is probably safe during the later stages of pregnancy, their systemic absorption is higher than when applied to the skin. The systemic antifungal drug with which there has been the most experience in pregnancy is amphotericin B. There have been no reports of teratogenesis attributed to this agent. There is evidence to suggest that fluconazole exhibits dose-dependent teratogenic effects; however, it appears to be safe at lower doses (150 mg/day). Ketoconazole, flucytosine, and griseofulvin have been shown to be teratogenic and/or embryotoxic in animals. Iodides have been associated with congenital goiter and should not be used during pregnancy.

Introduces efficient pipeline architectures for the recursive morphological operations. The standard morphological operation is applied directly on the original input image and produces an output image. The order of image scanning in which the operator is applied to the input pixels is irrelevant. However, the intent of the recursive morphological operations is to feed back the output at the current scanning pixel to overwrite its corresponding input pixel to be considered into computation at the following scanning pixels. The resultant output image by recursive morphology inherently depends on the image scanning sequence. Two pipelined implementations of the recursive morphological operations are presented. The design of an application-specific systolic array is first introduced. The systolic array uses 3 n cells to process an nxn image in 6 n-2 cycles. The cell utilization rate is 100%. Second, a parallel program implementing the recursive morphological operations and running on distributed-memory multicomputers is described. Performance of the program can be finely tuned by choosing appropriate partition parameters.

Restricting the NaCl content in the rat maternal and preweaning diet results in a significant and specific reduction (60%) of chorda tympani nerve responsiveness to sodium stimuli in the offspring. Repletion of dietary sodium at any time during postnatal development results in a complete and persistent recovery of chorda tympani nerve function. To learn whether the maturation of postsynaptic cells are also affected by the early dietary manipulation, dendritic morphology, neuronal and glial densities and numbers were studied within the area of the nucleus of the solitary tract (NTS). Examination of dendritic morphologies in Golgi-Cox stained neurons revealed that cells with multipolar and fusiform somata in the rostral NTS exhibited longer dendrites following dietary NaCl deprivation during development (deprived rats) than in controls. These changes were generally maintained in rats initially deprived of NaCl and then fed a NaCl-replete diet postweaning ("recovered" rats). In contrast, ovoid neurons were not affected by NaCl deprivation but had increases in the lengths of their dendrites following "recovery." Along with dendritic alterations, the packing density of neurons in the rostral NTS was greater in NaCl-deprived rats than in controls, but was similar to controls following "recovery." Glial packing density also increased following deprivation and remained high in "recovered" rats. These results indicate that activity-dependent events as well as events not related to afferent activity (e.g., hormonal changes) may influence the morphological development of NTS neurons. In addition, significant interactions among primary afferent fibers, central neurons, and glia may direct development within the central gustatory system.

As the case presented here illustrates, nocardiosis, like other infections in which cell-mediated immunity plays a large defensive role, can relapse after apparent cure and occasionally at times remote from the original infection. Although relapse in patients with transplants has been cited as a reason for continued prophylaxis, only a few of these cases are adequately documented. This case supports the advice of those authors who give suppressive antibiotic therapy for the duration of immunosuppression in transplant recipients recovering from infections due to Nocardia sp. Alternatively, many transplant centers are routinely using TMP/SMX chemoprophylaxis in all solid organ transplantations to prevent opportunistic infections with Pneumocystis and Listeria sp. Primary prophylaxis has also been associated with a decreased incidence of nocardial infections.

A case of fatal disseminated fungal infection due to Conidiobolus coronatus in a patient with a renal transplant is described. This organism, known to cause localized infections in otherwise healthy individuals in the tropics, is now recognized as a cause of fatal infection in immunosuppressed hosts. Histologically, localized infections are characterized by lack of vessel invasion and the presence of an eosinophilic sleeve around fungal elements, called the Splendore-Hoeppli phenomenon. The histologic findings in the present case were more typical of mucormycosis, and the correct diagnosis was established only after the organism was isolated and identified in culture.

In order to determine whether the developing central gustatory system responds to altered sensory experience, terminal fields of the chorda tympani nerve (CT) within the nucleus of the solitary tract (NTS) in control, NaCl deprived, and rats in which CT taste responses "recovered" from NaCl deprivation were investigated via anterograde transport of HRP. Rats fed a low sodium diet (0.03% NaCl) from the third day of gestation to at least 35 days postnatal exhibited both abnormally distributed and irregularly shaped CT terminal fields. Specifically, the dorsal zone of the field was the smallest in controls whereas it was the largest in deprived rats, occupying more medial and caudal territory within the nucleus. The portion of the field immediately ventral to the dorsalmost zone was characterized by a compact, oval shape in control rats and an irregular, broad configuration in deprived rats. Although it has been observed that deprivation-induced changes in the neurophysiology of the CT are reversible, the central morphological alterations reported here remain abnormal. Restoration of 1.0% NaCl in the diet at 28 days postnatally, for at least 60 days, did not result in normal CT terminal fields. The pattern of the field in rats "recovered" from NaCl deprivation was comparable to that found in deprived rats, and the size of the field was three times that found in control and deprived rats. The terminal fields of another nerve containing gustatory afferents, the lingual-tonsilar branch of the glossopharyngeal nerve (LT-IX), were studied for comparison. Interestingly, the pattern of the LT-IX field was not altered by sodium deprivation. The relative size and topography of the LT-IX fields in deprived rats were similar to controls. Thus, sodium deprivation appears to alter selectively the anatomical organization of the CT. Differences in vulnerability between the CT and LT-IX terminal fields may derive from differences in the responsiveness of these nerves to NaCl, and/or to differences in the timing of early neural events.

Benzomorphan interactions with acetylcholine receptor complexes from Torpedo californica electric organ were investigated using radiolabelled probes. All of the benzomorphans had minimal affinity for the acetylcholine binding site. Four benzomorphans (N-allylnormetazocine, cyclazocine, phenazocine and pentazocine) inhibited the binding of tritiated perhydrohistrionicotoxin and phencyclidine to ion channel sites with IC50 values between 0.4 and 5 microM. Two other benzomorphans, ketazocine and ethylketocyclazocine, were much less active. Thus, ion channel affinity is not limited to benzomorphans which stimulate sigma-opiate receptors, and is not a certain indication of psychotomimetic potency.

The specific red cell adherence test as a method to detect blood group antigen deletion in urothelial malignancy has been reported to yield approximately 40 per cent false negative results in 0 blood group patients. Our study of multiple sections of 8 normal ureters from blood group 0 patients and more than 220 specimens of transitional cell cancer taken from 48 patients reveals that the immunoperoxidase technique is more specific than the specific red cell adherence method in predicting subsequent invasion in blood group O(H) patients presenting with superficial transitional cell carcinomas (71 compared to 29 per cent) but is no more specific for tumors containing A or B antigens. However, immunoperoxidase staining does improve discernment of underlying histologic detail and, thereby, facilitates recognition of false positive antigen testing associated with squamous and adenomatous metaplasia. Areas of squamous and adenomatous metaplasia in specimens we tested were frequently antigen positive in invasive tumors. Therefore, we believe that these areas must be disregarded in determining antigen deletion in transitional cell carcinomas.

A new sequence-specific endonuclease, SfaI, has been partially purified from Streptococcus faecalis subsp. zymogenes. SfaI recognizes the tetranucleotide sequence 5'G-G-C-C 3' 3' C-C-G-G 5' and cleaves it at the sites indicated by the arrows.

Specimens of transitional cell carcinoma of the ureter and renal pelvis from 20 patients were tested for blood group antigenicity using immunoperoxidase and specific red cell adherence methods. The results of antigen testing were correlated with tumor stage and grade as well as the subsequent clinical course of the patients. The specific red cell adherence test was negative in 80 per cent and the immunoperoxidase test was negative in 40 per cent of all tumors. Of the 4 patients with positive specific red cell adherence tests 3 had high grade (II to III), invasive tumors as did 7 of 12 with tumors that were positive by immunoperoxidase testing. Blood group antigen testing did not prove helpful in predicting the clinical course of our patients. In addition, a careful review of previously published data does not support the conclusion that blood group antigen testing is a valuable predictor of upper tract tumor aggressiveness.

Chlorcyclizine and structurally related drugs induce a high incidence of cleft palate and skeletal malformations in fetal rats. We have shown previously that these teratogens bind tightly and reversibly to chondroitin sulfate of cartilage and compete with calcium for binding. Experiments reported here demonstrate that co-administration of calcium chelating agents with chlorcyclizine significantly increases both the frequency of malformations and retention of [14C] chlorcyclizine by embryos. Retention of radioactive teratogen by embryos is inverse to retention of [45Ca]calcium. These findings suggest that drug binding to embryonic glycosaminoglycans is involved in the pathogenesis of malformations produced by chlorcyclizine.

Administration of the cleft palate teratogen chlorcyclizine or norchlorcyclizine to pregnant rats causes an alteration in glycosaminoglycans (GAGs) in embryonic palatal shelves. Pulse-chase experiments in vitro indicate that norchlorcyclizine enhances the degradation of hyaluronic acid and chondroitin sulfate but has little or no effect on their synthesis. These changes in GAGs are caused by concentrations of norchlorcyclizine that have no appreciable effect on DNA or protein synthesis. These findings suggest that degradation of palatal GAGs may be the primary biochemical defect responsible for the inhibition of palatal shelf elevation by norchlorcyclizine.

Histrionicotoxin, a spiropiperidine alkaloid, and twenty-two analogs inhibited binding of [3H]perhydrohistrionicotoxin [( 3H]H12-HTX) and of [3H]phencyclidine [( 3H]PCP) to sites on the acetylcholine receptor-ion complex of Torpedo electroplax membranes. Structural alterations to the nitrogen (secondary amine) or oxygen (alcohol) functions or to the five carbon and four carbon side chain of histrionicotoxin altered the potency versus [3H]H12-HTX and [3H]PCP binding measured in the presence or absence of a receptor agonist, carbamylcholine. Histrionicotoxin itself was 3-fold more potent versus [3H]PCP binding than versus [3H]H12-HTX binding. N-Methylation or O-acetylation increased this difference, while alterations to the side chains either slightly decreased or markedly increased this difference. Histrionicotoxin was some 3.5-fold more potent versus [3H]H12-HTX binding in the presence of carbamylcholine than in its absence. O-Acetylation increased this selectivity for the carbamylcholine-activated state of the receptor channel complex, while alterations in the side chains either reduced or increased the selectivity. Histrionicotoxin was some 2.2-fold more potent versus [3H]PCP binding in the presence of carbamylcholine than in its absence. N-Methylation of O-acetyl-histrionicotoxin greatly increased this selectivity, while alterations in the side chains either reduced or had no effect on selectivity.