Ulceroglandular tularemia.

A 14-year-old boy presented with fevers and nonspecific flu-like symptoms, as well as an enlarging ulcerated plaque involving the upper back, lymphadenopathy, and bilateral pulmonary nodules. Bacterial cultures of ulcer tissue grew Francisella tularensis on enriched chocolate agar plates. Making the diagnosis requires a high index of suspicion, and communication with the laboratory to successfully and safely culture these highly pathogenic bacteria is imperative.

Eliapixant is a selective P2X3 receptor antagonist for the treatment of disorders associated with hypersensitive nerve fibers.

ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.

Primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma.

Cutaneous T-cell lymphomas most commonly have a CD4(+) memory T-cell phenotype with relatively indolent course, but may in rare cases present with a CD8(+) cytotoxic phenotype exhibiting strikingly more aggressive clinical behavior. We present two cases of the clinically aggressive subtype of primary cutaneous epidermotropic CD8(+) cutaneous T-cell lymphoma and review the current literature, clinical behavior, and recommendations for treatment distinct from that of more common CD4(+) variants of cutaneous T-cell lymphoma.

Development of the predictor HERG fluorescence polarization assay using a membrane protein enrichment approach.

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

Human CD4+ effector T cells mediate indirect interleukin-12- and interferon-gamma-dependent suppression of autologous HLA-negative lung tumor xenografts in severe combined immunodeficient mice.

A human/severe combined immunodeficient mouse chimeric model was used to demonstrate that peripheral blood leukocytes (PBLs) from a patient with lung cancer completely suppress the growth of an autologous tumor in a PBL dose-dependent fashion repeatedly and over a 4-year period. Suppression of the patient's tumor required CD4+ T cells, CD56+ natural killer cells, and CD14+ monocytes/macrophages, but was completely independent of CD8+ T cells. The CD4+ effector cells promoted tumor killing indirectly because direct tumor recognition and killing are precluded by the absence of MHC class I and II molecules on the tumor cells. Tumor suppression was found to require both human interleukin-12 (IL-12) and IFN-gamma, which were produced and released by the patient's monocytes and T cells, respectively. These results establish that human CD4+ T cells present in the peripheral blood of a patient with lung cancer are able to orchestrate cytokine-dependent killing of an autologous MHC-negative tumor indirectly and without codependence on CD8+ T cells. We conclude that human tumor suppression is achieved in vivo even in the absence of MHC molecules on tumor cells. This tumor suppression is mediated indirectly by cytokines produced by the patient's PBLs that ultimately initiate tumor killing via several, presently incompletely defined mechanisms.

Association of change in clinical status and change in the percentage of the CD4+CD26- lymphocyte population in patients with Sézary syndrome.

BACKGROUND: Because there are currently many effective therapies available for Sézary syndrome, close monitoring of disease progression is required in order for a clinician to know when to institute or change an intervention. It has been our clinical experience that changes in patients' CD4+CD26- T-cell populations of peripheral blood lymphocytes herald changes in their clinical status. OBJECTIVE: Our purpose was to evaluate whether a change in patients' CD4+CD26- population of T cells presages a change in their clinical status. We also sought to investigate the association between a change in T-cell populations that are CD4+CD7-, CD8+, CD56+, and the CD4+/CD8+ T-cell ratio and a change in the patient's clinical status. METHODS: We conducted a retrospective chart review analysis of 21 patients with Sézary syndrome who had flow cytometry, usually including levels of CD4+CD26-, CD4+CD7-, CD8+, CD56+, and CD4+/CD8+ ratios measured at two time periods, 12 weeks apart. RESULTS: We report two cases in which changes in patients' clinical status were preceded by several weeks by a change in their CD4+CD26- level. We report weak associations between a decreasing CD4+CD26- T-cell population, a decreasing CD4+CD7- population, an increasing CD56+ population, and an improving clinical status. We also report stronger associations between both a decreasing CD8+ population and an increasing CD4+/CD8+ ratio and a worsening clinical status. LIMITATIONS: The study was limited by the number of patients and the time period over which the study was conducted. In addition, varying configurations of CD4+CD26- T-cell populations were observed that may have limited the utility of this measurement. CONCLUSIONS: Flow cytometry assays of patients' blood and, in particular, measurement of the CD4+CD26- population of lymphocytes over time may be a valuable tool for monitoring patients with Sézary syndrome. There exist varying configurations of CD26 T lymphocytes that may cause differences in standards for what is considered positive and negative between observers. Further prospective analysis involving larger groups of patients is recommended.

Recent advances in the modulation of voltage-gated ion channels for the treatment of epilepsy.

Regardless of the voltage-gated ion channel that is targeted in a drug discovery effort for the treatment of epilepsy, two routes have been followed historically: 1). a compound initially, and often surreptitiously, discovered due to activity in animal seizure models is further optimized by medicinal chemistry, or 2). a molecular target is identified based on the phenotype of transgenic animals, or linkage studies from humans with the disease, and compounds are then investigated within a mechanistic framework. Antagonists of voltage-gated sodium channels have been pursued utilizing primarily the first approach; many of these compounds also have significant activity at other ion channels. Both approaches have been utilized to discover voltage-gated calcium channel antagonists, although most efforts to date have used the first approach. Several spontaneous mutant mice and transgenic animals have been utilized to probe the role of the numerous voltage-gated calcium channel subunits and their isoforms as potential molecular targets. Compounds that open or prolong the opening of voltage-gated potassium channels have been discovered using the first approach, with a detailed understanding of the molecular target and mechanism of action coming to light several years later. Genetic evidence from humans is limited to relatively rare forms of epilepsy, and transgenic animals with interesting phenotypes do not always translate into good molecular targets in humans. No clinically-useful antiepileptic drug (AED) has been developed to date that specifically interacts with one, or even one class, of ion channels to produce a therapeutic effect. The tools now exist to search for potent, selective, and safe ion channel modulators for the treatment of epilepsy. This review seeks to summarize the most recent pre-clinical and clinical efforts focused on voltage-gated ion-channels for the development of AEDs.

SCID mouse models to study human cancer pathogenesis and approaches to therapy: potential, limitations, and future directions.

The successful engraftment of human tumors and human immunocompetent cells into severe combined immunodeficient (SCID) mice has led to the generation of a wide array of different experimental designs that have proven useful in studying the cell biology of human cancer, and for evaluating novel therapeutic approaches to the treatment of cancer. In this review five of the most frequently used embodiments of the SCID model are presented. The goals of this review are to discuss how each model has been utilized to study human cancer and its response to many different novel therapies, to provide an assessment of the strengths and limitations of each model, and to outline future directions with a focus on what is needed to overcome some of the current limitations and pitfalls of the SCID models.

The importance of being profiled: improving drug candidate safety and efficacy using ion channel profiling.

Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008).

Uncertainty in the perioperative management of high-risk cutaneous squamous cell carcinoma among Mohs surgeons.

OBJECTIVE: To evaluate whether Mohs surgeons' management of high-risk cutaneous squamous cell carcinoma (HRCSCC) is uniform regarding radiologic nodal staging (RNS) and adjuvant radiation therapy (ART). DESIGN: A survey study of randomly selected, fellowship-trained Mohs surgeons. SETTING: An academic medical center. PARTICIPANTS: American College of Mohs Surgery members who responded to an e-mail invitation completed either a survey regarding management of HRCSCC (n=117) or SCC with perineural invasion (PNI) (n=118). Participants totaled approximately 25% of the American College of Mohs Surgery membership. MAIN OUTCOME MEASURES: (1) Percentage of patients with HRCSCC referred for RNS, sentinel lymph node biopsy (SLNB), or ART over the preceding 12 months; (2) top factors leading surgeons to consider RNS, SLNB, or ART; and (3) acceptance of ART for clinical scenarios of various degrees of PNI. RESULTS: Most respondents cited PNI and in-transit metastasis as top factors leading to consideration of RNS, SLNB, or ART. Otherwise, there was no consensus regarding use of, or indications for, RNS, SLNB, or ART. CONCLUSIONS: The lack of consistency between experts indicates that there is equipoise regarding indications for RNS and ART in HRCSCC. There is also wide variation in RNS and ART practices among Mohs surgeons who are specifically trained to manage HRCSCC. Clinical trials should therefore be conducted in these areas as there is no clear standard of care.

IL-21 enhances antitumor responses without stimulating proliferation of malignant T cells of patients with Sézary syndrome.

IL-21, a common gamma-chain cytokine secreted by activated CD4+ T cells, influences both humoral and cell-mediated immune responses through the regulation of T, B, dendritic, and natural killer (NK) cells. Sézary syndrome is an advanced form of cutaneous T-cell lymphoma, a clonally derived malignancy of CD4+ T cells that is characterized by profound defects in host cellular immune function. As a modulator of both innate and adaptive immune responses, IL-21 could play an important role in augmenting cell-mediated immunity in these patients. Normal donor and Sézary syndrome patient peripheral blood mononuclear cells were cultured with IL-21 and tested for CD8+ T- and NK-cell activation, NK-cell cytotoxicity, and tumor cell proliferation and apoptosis. IL-21 resulted in a modest increase in CD8+ T- and NK-cell activation, associated with a marked increase in cytolytic activity against both K562 and malignant CD4+ T-cell targets. Although IL-21 failed to demonstrate pro-apoptotic effects on the malignant CD4+ T cells, it is noteworthy that it had no demonstrable proliferative effects on these cells. Thus, IL-21 may play an important role in enhancing the host immune response of Sézary syndrome patients through the increased cytolytic activity of T and NK cells.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma.

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.