Elucidating the molecular basis of action of a classic drug: guanidine compounds as inhibitors of voltage-gated potassium channels

Guanidine and its alkyl analogs stimulate the neuromuscular junction presynaptically by inhibiting voltage-gated potassium (Kv) channels, leading to enhanced release of acetylcholine in the synaptic cleft. This stimulatory effect of guanidine underlies its use in the therapy for the neuromuscular diseases myasthenic syndrome of Lambert-Eaton and botulism. The therapeutic use of guanidine is limited, however, because of side effects that accompany its administration. Therefore, the design of guanidine analogs with improved therapeutic indices is desirable. Progress toward this goal is hindered by the lack of knowledge of the mechanism by which these molecules inhibit Kv channels. Here we examine an array of possible mechanisms, including charge screening, disruption of the protein-lipid interfaces, direct interaction with the voltage sensors, and pore-binding. Our results demonstrate that guanidines bind within the intracellular pore of the channel and perturb a hydrophobic subunit interface to stabilize a closed state of the channel. This mechanism provides a foundation for the design of guanidine analogs for the therapeutic intervention of neuromuscular diseases.     

Modeling prostate cancer: a perspective on transgenic mouse models

Despite considerable success in treatment of early stage localized prostate cancer (PC), acute inadequacy of late stage PC treatment and its inherent heterogeneity poses a formidable challenge. Clearly, an improved understanding of PC genesis and progression along with the development of new targeted therapies are warranted. Animal models, especially, transgenic immunocompetent mouse models, have proven to be the best ally in this respect. A series of models have been developed by modulation of expression of genes implicated in cancer-genesis and progression; mainly, modulation of expression of oncogenes, steroid hormone receptors, growth factors and their receptors, cell cycle and apoptosis regulators, and tumor suppressor genes have been used. Such models have contributed significantly to our understanding of the molecular and pathological aspects of PC initiation and progression. In particular, the transgenic mouse models based on multiple genetic alterations can more accurately address the inherent complexity of PC, not only in revealing the mechanisms of tumorigenesis and progression but also for clinically relevant evaluation of new therapies. Further, with advances in conditional knockout technologies, otherwise embryonically lethal gene changes can be incorporated leading to the development of new generation transgenics, thus adding significantly to our existing knowledge base. Different models and their relevance to PC research are discussed.     

水体中克雷伯菌属的分布及其在水源性急性肠道感染中的价值

俄罗斯不同气候地区不同功能水体中克雷伯菌属广泛分布。克雷伯菌属可见于遭受生物、化学污染的集中供水的地表水源,无防护的地下蓄水层,缺乏有效清洁、消毒系统的饮用水。研究表明,水体中的克雷伯菌属具有致病性和毒性,对现代药物和消毒剂(氯、紫外线)具有抗性,很容易穿透进入地下蓄水层。克雷伯菌属细菌有很强的致病性(粘附力、侵袭力、磷酸酯酶、卵磷脂酶、脱氧核糖核酸酶、溶血活性),含有致病性遗传标记cnf-1。克雷伯菌属(100 CFU/dm³)可引起急性肠道感染。在不检测总大肠菌群的情况下,检测水体尤其是饮用水中的克雷伯菌属,可以评估所用水的流行病学危险。     

Robot-assisted radiofrequency ablation of primary and secondary liver tumours: early experience

Objective

Computed tomography (CT)-compatible robots, both commercial and research-based, have been developed with the intention of increasing the accuracy of needle placement and potentially improving the outcomes of therapies in addition to reducing clinical staff and patient exposure to radiation during CT fluoroscopy. In the case of highly inaccessible lesions that require multiple plane angulations, robotically assisted needles may improve biopsy access and targeted drug delivery therapy by avoidance of the straight line path of normal linear needles.

Methods

We report our preliminary experience of performing radiofrequency ablation of the liver using a robotic-assisted CT guidance system on 11 patients (17 lesions).

Results/Conclusion

Robotic-assisted planning and needle placement appears to have high accuracy, is technically easier than the non-robotic-assisted procedure, and involves a significantly lower radiation dose to both patient and support staff.

Key Points

? An early experience of robotic-assisted radiofrequency ablation is reported ? Robotic-assisted RFA improves accuracy of hepatic lesion targeting ? Robotic-assisted RFA makes the procedure technically easier with significant lower radiation dose     

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141⁺ DC subset. CD141⁺ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c⁺ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141⁺ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8⁺ cytotoxic T lymphocytes than poly I:C–activated CD1c⁺ DCs. Importantly, CD141⁺ DCs, but not CD1c⁺ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141⁺ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α⁺ DC subset. The data demonstrate a role for CD141⁺ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.The essential role of DCs in the induction and regulation of immune responses to pathogens, self-antigens (Ags), and cancers is now well established. All DCs excel at processing and presenting Ag and priming naive T cell responses, but the complexity of DC subsets and their individual specialized functions is just becoming apparent (MacDonald et al., 2002; Villadangos and Schnorrer, 2007; Naik, 2008). Promising DC-based therapeutic vaccines have been described to treat malignancies and infections (Vulink et al., 2008), but the majority of these use in vitro–generated monocyte-derived DC (MoDC), and the physiological standing of this DC subtype is currently unclear. Understanding the emerging complexities of human DC subset biology is therefore essential to develop new vaccines and therapeutics targeting DC.The characterization and function of human DC subsets has been confounded by their rarity, the lack of distinctive markers, and limited access to human tissues. Human blood DCs comprise ∼1% of circulating PBMCs and have been classically defined as Ag-presenting leukocytes that lack other leukocyte lineage markers (CD3, 14, 15, 19, 20, and 56) and express high levels of MHC class II (HLA-DR) molecules (Hart, 1997). These can be broadly categorized into two groups: plasmacytoid CD11c⁻CD123⁺ DC and conventional or myeloid CD11c⁺CD123⁻ DC. We have described three further phenotypically distinct subsets of CD11c⁺ DC, defined by their expression of CD16, CD1c (BDCA-1), and CD141 (BDCA-3; MacDonald et al., 2002). Gene expression profiling and hierarchical clustering data has indicated that plasmacytoid DC and CD16⁺ DC arise from separate precursor cells, whereas the CD1c⁺ DC and CD141⁺ DC subsets appear to have a common origin and represent two different stages of a similar subset (Lindstedt et al., 2005). However, CD1c⁺ and CD141⁺ DCs each have unique gene expression profiles distinct from monocytes and MoDC, and this predicts that they have different functions (Dzionek et al., 2000; MacDonald et al., 2002; Lindstedt et al., 2005).The concept of distinct DC subtypes with unique capabilities to influence immunological outcomes is exemplified by the mouse CD8α⁻ and CD8α⁺ conventional DC subsets that reside in the lymph nodes and spleen (Villadangos and Schnorrer, 2007; Naik, 2008). The CD8α⁻ DC subset appears to be most effective at inducing Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999) and processing and presenting Ag to CD4⁺ T cells via the MHC class II pathway (Pooley et al., 2001; Dudziak et al., 2007; Villadangos and Schnorrer, 2007). In contrast, the CD8α⁺ DC subset has a unique ability to take up dead or dying cells and to process and present exogenous Ag on MHC class I molecules to CD8⁺ T cells (i.e., cross-presentation; den Haan et al., 2000; Iyoda et al., 2002; Schnorrer et al., 2006). There is now substantial evidence that the CD8α⁺ DC subset plays a crucial role in the induction of protective CD8⁺ CTL responses that are essential for the eradication of cancers, viruses, and other pathogenic infections (Dudziak et al., 2007; Hildner et al., 2008; López-Bravo and Ardavín, 2008; Naik, 2008). The identification of the human DC subset with similar functional capacity would be a significant advance and would enable translation of mouse DC biology into clinical practice.Correlation of the human and mouse DC subsets has been hampered by differences in their defining markers (human DCs do not express CD8α). Interestingly, computational genome-wide expression profiling clustered human CD141⁺ DC and CD1c⁺ DC with the mouse CD8α⁺ and CD8α⁻ conventional DC subsets, respectively (Robbins et al., 2008). Human CD141⁺ DC and mouse CD8α⁺ DC share a number of phenotypic similarities, including expression of Toll-like receptor (TLR) 3 (Edwards et al., 2003; Lindstedt et al., 2005), the novel surface molecule Necl2 (nectin-like protein 2; Galibert et al., 2005), and the C-type lectin CLEC9A (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). Thus, whether the human CD141⁺ DC subset is the human functional equivalent of the mouse CD8α⁺ DC subset has now become a major question for immunologists.CD141⁺ DCs constitute only ∼0.03% of human PBMCs and, although present in other human tissues, their low proportions and difficulties with aseptic human tissue access mean that they have never been isolated in sufficient quantity to study their function until now. We report the first detailed functional analysis of human CD141⁺ DCs in response to TLR3 stimuli and define their role in the induction of Th1 responses and cross-presentation.     

Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1′ domain, and using genetically modified mice that contain the human M1′ domain inserted into the mouse IgE locus, we demonstrated that M1′-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1′-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1′-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1′-specific antibodies may be a novel treatment for asthma and allergy.     

Response of fractured osteoporotic bone to polymethylmethacrylate after vertebroplasty: case report

BACKGROUND CONTEXT: Polymethylmethacrylate (PMMA) is the most commonly used bone cement for vertebroplasties to treat osteoporotic vertebral compression fractures (VCFs). Several studies have described the reaction of normal bone to PMMA, but it is still unclear how fractured osteoporotic bone responds to PMMA. PURPOSE: To describe the response of fractured osteoporotic bone to PMMA after vertebroplasty. STUDY DESIGN/SETTING: Case report. METHODS: A 69-year-old woman with a previous vertebroplasty at T8 to treat an osteoporotic VCF was admitted to the hospital after she developed lower extremity motor weakness, diffuse hypoesthesia and decreased rectal tone. Magnetic resonance imaging studies of the thoracic spine showed that she had severe spinal cord compression at the level of T8 and T9, as well as akyphotic deformity. A corpectomy of T8 and T9 was performed as part of a spinal cord decompression procedure. Tissue from vertebral body T8, intervertebral discs T7-T8 and T8-T9 and the PMMA implant were then submitted for histologic evaluation.RESULTS: Vertebral body T8 demonstrated viable bone trabeculae, osteoid. fibrosis, granulation tissue and multinucleated giant cells containing PMMA. Scattered necrotic bone fragments were identified throughout the vertebral body, most evident near the PMMA. PMMA leakage into the T7-T8 disc was identified without significant disc inflammation or necrosis. CONCLUSION: Fractured osteoporotic bone is capable of undergoing a reparative healing response after vertebroplasty using PMMA.     

Epidemiology of persistent diarrhea among Guatemalan rural children

A prospective, longitudinal two-year study to determine the epidemiology of persistent ( 14 days'duration) diarrhea in rural children of Guatemala was undertaken. Three-hundred and twenty-one children aged 0-35 months were kept under surveillance by twice-a-week home visits. The overall incidence of diarrhea was 0.147 per child-week; the incidence of persistent diarrhea was 0.014 per child-week. The peak of persistent diarrhea was observed in infants below six months of age, with a continuous decline thereafter. This trend in incidence of persistent diarrhea was associated with a higher proportion (16%) of illnesses persisting for more than 13 days in children younger than six months of age as compared to children 30-35 months old (4%). Males had more diarrhea (0.156 per child-week) than females (0.139 per child week). Among children above 18 months of age, the proportion of episodes that lasted for more than 13 days was lower in females than in males.     

Autoerythrocyte sensitization syndrome (Gardner–Diamond syndrome): review of the literature**

A review of the literature concerning psychogenic purpura is presented. The diagnosis is usually based on typical anamnestic data, clinical presentation (painful inflammatory skin lesions, which progressed to ecchymoses during the next 24 h) and positive diagnostic tests with intracutaneous injections of 80% solution of washed autologous erythrocytes. No pathological findings of blood coagulation parameters are usually detected. Histopathological evaluations of lesional biopsies revealed non-specific changes. Taking into account the high frequency of psychic disorders and stress dependence of skin symptoms, therapy with psychotropic drugs (according to indications) and psychotherapy are pathogenetically grounded methods of treatment in psychogenic purpura, and should be provided together with symptomatic therapy.