Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand

<p>Abstract</p> <p>Objective</p> <p>To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.</p> <p>Experimental Design</p> <p>The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. <it>In vivo </it>tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with <it>ex vivo </it>imaging.</p> <p>Results</p> <p>PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The <it>in vivo </it>and <it>ex vivo </it>optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.</p> <p>Conclusions and Clinical Relevance</p> <p>PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.</p

Differentiation and Recruitment of Th9 Cells Stimulated by Pleural Mesothelial Cells in Human Mycobacterium tuberculosis Infection

Newly discovered IL-9–producing CD4+ helper T cells (Th9 cells) have been reported to contribute to tissue inflammation and immune responses, however, differentiation and immune regulation of Th9 cells in tuberculosis remain unknown. In the present study, our data showed that increased Th9 cells with the phenotype of effector memory cells were found to be in tuberculous pleural effusion as compared with blood. TGF-β was essential for Th9 cell differentiation from naïve CD4+ T cells stimulated with PMA and ionomycin in vitro for 5 h, and addition of IL-1β, IL-4 or IL-6 further augmented Th9 cell differentiation. Tuberculous pleural effusion and supernatants of cultured pleural mesothelial cells were chemotactic for Th9 cells, and this activity was partly blocked by anti-CCL20 antibody. IL-9 promoted the pleural mesothelial cell repairing and inhibited IFN-γ-induced pleural mesothelial cell apoptosis. Moreover, pleural mesothelial cells promoted Th9 cell differentiation by presenting antigen. Collectively, these data provide new information concerning Th9 cells, in particular the collaborative immune regulation between Th9 cells and pleural mesothelial cells in human M. tuberculosis infection. In particular, pleural mesothelial cells were able to function as antigen-presenting cells to stimulate Th9 cell differentiation

The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats

Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A

Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/β-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent

Clara Cell 10-kDa Protein Gene Transfection Inhibits NF-κB Activity in Airway Epithelial Cells

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation

Effectiveness of manual therapy compared to usual care by the general practitioner for chronic tension-type headache: design of a randomised clinical trial

<p>Abstract</p> <p>Background</p> <p>Patients with Chronic Tension Type Headache (CTTH) report functional and emotional impairments (loss of workdays, sleep disturbances, emotional well-being) and are at risk for overuse of medication. Manual therapy may improve symptoms through mobilisation of the spine, correction of posture, and training of cervical muscles.</p> <p>We present the design of a randomised clinical trial (RCT) evaluating the effectiveness of manual therapy (MT) compared to usual care by the general practitioner (GP) in patients with CTTH.</p> <p>Methods and design</p> <p>Patients are eligible for participation if they present in general practice with CTTH according to the classification of the International Headache Society (IHS).</p> <p>Participants are randomised to either usual GP care according to the national Dutch general practice guidelines for headache, or manual therapy, consisting of mobilisations (high- and low velocity techniques), exercise therapy for the cervical and thoracic spine and postural correction. The primary outcome measures are the number of headache days and use of medication. Secondary outcome measures are severity of headache, functional status, sickness absence, use of other healthcare resources, active cervical range of motion, algometry, endurance of the neckflexor muscles and head posture. Follow-up assessments are conducted after 8 and 26 weeks.</p> <p>Discussion</p> <p>This is a pragmatic trial in which interventions are offered as they are carried out in everyday practice. This increases generalisability of results, but blinding of patients, GPs and therapists is not possible.</p> <p>The results of this trial will contribute to clinical decision making of the GP regarding referral to manual therapy in patients with chronic tension headache.</p

Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature

Cell–cell and cell–matrix dynamics in intraperitoneal cancer metastasis

The peritoneal metastatic route of cancer dissemination is shared by cancers of the ovary and gastrointestinal tract. Once initiated, peritoneal metastasis typically proceeds rapidly in a feed-forward manner. Several factors contribute to this efficient progression. In peritoneal metastasis, cancer cells exfoliate into the peritoneal fluid and spread locally, transported by peritoneal fluid. Inflammatory cytokines released by tumor and immune cells compromise the protective, anti-adhesive mesothelial cell layer that lines the peritoneal cavity, exposing the underlying extracellular matrix to which cancer cells readily attach. The peritoneum is further rendered receptive to metastatic implantation and growth by myofibroblastic cell behaviors also stimulated by inflammatory cytokines. Individual cancer cells suspended in peritoneal fluid can aggregate to form multicellular spheroids. This cellular arrangement imparts resistance to anoikis, apoptosis, and chemotherapeutics. Emerging evidence indicates that compact spheroid formation is preferentially accomplished by cancer cells with high invasive capacity and contractile behaviors. This review focuses on the pathological alterations to the peritoneum and the properties of cancer cells that in combination drive peritoneal metastasis