Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

In this study, we found that a subpopulation of CD4(+)CD25(+) (85% Foxp3(+)) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4(+)CD25(+) (85% Foxp3(+)) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+C)D25(+)Foxp3(+)D4GDI(+) cells do not produce IL-10, TGF-ß and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1ß, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+)) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+)CD25(+) (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

Illinois Breast and Cervical Cancer Program: Implementing Effective Public-Private Partnerships to Assure Population Health.

With the implementation of the Affordable Care Act (ACA), it is essential for the public health sector to elucidate its role with respect to its mission of assuring population health, and to clarify its role with respect to the private health care system. To that end, we examined the value added to the population health enterprise of successful public-private partnerships (PPPs) such as those found in the Illinois Breast and Cervical Cancer Program (IBCCP), the Centers for Disease Control and Prevention's (CDC's) National Breast and Cervical Cancer Early Detection Program (NBCCEDP) in Illinois. Key Informant (KI) interviews focused on IBCCP implementation were conducted with IBCCP lead agency (LA) program coordinators (n = 35/36) in winter 2012-2013. Analysis was conducted using Atlas.ti software. The KI interviews revealed the existence of highly developed PPPs between the IBCCP LAs and individual medical providers and hospitals across Illinois. The data suggest that the small amount of funding provided by IBCCP to each LA in Illinois has been used to build and sustain robust PPPs in the majority of the IBCCP communities. The PPPs developed through the IBCCP can be seen as an unplanned benefit of CDC's investment in breast and cervical health through the NBCCEDP. While the IBCCP/NBCCEDP might be considered a "boutique" categorical program which some may consider no longer necessary as individuals gain insurance under the ACA, the KI data underscore the critical role of public sector dollars, not only to serve individuals and communities directly but also to mobilize the private health care sector to act in partnership with public entities and become advocates for underserved communities.

Brainstem tuberculoma in the immunocompetent: case report and literature review.

Brainstem tuberculoma is an uncommon entity especially in the immunocompetent adult. The authors describe the case of a 32-year-old immunocompetent woman with no systemic signs or symptoms of tuberculosis, presenting with sudden-onset hemiparesis and diplopia. MRI of brain revealed an enhancing lesion in the midbrain and thalamus. Cerebrospinal fluid examination was normal. Chest imaging was consistent with miliary tuberculosis; however initial expectorated sputum was culture negative. Bronchosopy sputum culture was positive for Mycobacterium tuberculosis. Her PPD was negative. Patient became asymptomatic with treatment for tuberculosis and follow-up MRI showed complete resolution of the lesion a year later. We conclude that a high index of suspicion is essential to make an early diagnosis of intracranial tuberculoma, since often, traditional TB tests like PPD and sputum examinations can be negative.

Identification of cripto-1 as a novel serologic marker for breast and colon cancer.

PURPOSE: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. EXPERIMENTAL DESIGN: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. RESULTS: Very low levels of CR-1 (mean+/-SD) were detected in the plasma of healthy volunteers (0.32+/-0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68+/-3.5 ng/mL) and in patients with breast carcinoma (2.97+/-1.48 ng/mL; P<0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7+/-0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P<0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. CONCLUSION: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling.

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

Human Cripto-1 overexpression in the mouse mammary gland results in the development of hyperplasia and adenocarcinoma.

Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals.

Role of human cripto-1 in tumor angiogenesis.

BACKGROUND: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. METHODS: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. RESULTS: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CONCLUSION: CR-1 appears to have an important role in the multistep process of angiogenesis.

Developing the first assistant course at the Countess of Chester Hospital.

First assistant or advanced scrub practitioner roles have been undertaken by nurses since Florence Nightingale. Recently, theatre practitioners have undertaken this role as a matter of course, regardless of skill level. The Countess of Chester Hospital Theatres has produced the IS3 110 operating theatre first assistant module at level three. This runs alongside an in-house pathway of rapid evaluation of competence. Both are undertaken as work-based learning.

Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from MMTV-Cripto-1 transgenic mice.

Epithelial-mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto-1 (CR-1), a member of the epidermal growth factor-CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene by the MMTV promoter (MMTV-CR-1) and in mouse mammary epithelial cell line HC-11 overexpressing CR-1 (HC-11/CR-1). Western blot analysis showed decreased expression of E-cadherin in MMTV-CR-1 tumors and in HC-11/CR-1 cells. The expression of N-cadherin, vimentin, cyclin-D1, and of the zinc-finger transcription factor, snail, was increased in MMTV-CR-1 tumors. Increased snail mRNA was also found in HC-11/CR-1 cells. Expression of phosphorylated (P)-c-Src, P-focal adhesion kinase (FAK), P-Akt, P-glycogen synthease kinase 3beta (GSK-3beta), dephosphorylated (DP)-beta-catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV-CR-1 tumors. Immunohistochemistry showed positive staining for vimentin, N-cadherin, cyclin-D1, smooth muscle actin, fibronectin, snail, and beta-catenin in MMTV-CR-1 tumor sections. HC-11/CR-1 cells treated with the c-Src inhibitor PP2 reduced the expression of P-c-Src and of P-FAK, P-Akt, P-GSK-3beta, DP-beta-catenin all known to be activated by c-Src. Migration of HC-11/CR-1 cells was also reduced by PP2 treatment. These results suggest that CR-1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.

Clinical and radiographic features of HIV-related tuberculosis.

Human immunodeficiency virus (HIV)-related tuberculosis can be life-threatening for the individual, transmissible to others, and difficult to diagnose. We review the clinical, radiographic, and histopathologic features of HIV-related tuberculosis, and the ways in which these features are affected by the degree of immunodeficiency. At CD4 cell counts greater than 350 cells/microL, HIV-related tuberculosis has a similar presentation to tuberculosis in HIV-uninfected adults, predominantly pulmonary involvement with fibronodular and/or cavitary infiltrates. With progressive immunodeficiency, extrapulmonary involvement becomes increasingly common. Pulmonary involvement remains common at all stages of HIV disease, but the radiographic pattern is very different among persons with advanced immunodeficiency, in whom the most common abnormalities are intrathoracic adenopathy, focal lower or middle lobe infiltrates, and diffuse miliary or nodular infiltrates. The keys to the diagnosis of HIV-related tuberculosis are knowledge of the epidemiology of tuberculosis, recognition of the ways that immunodeficiency changes the clinical presentation, and an assiduous effort to obtain specimens for mycobacterial smear and culture.

Identification and characterization of three immunodominant structural proteins of fowlpox virus.

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.