Proteomic Identification of Immunodiagnostic Antigens for Trypanosoma vivax Infections in Cattle and Generation of a Proof-of-Concept Lateral Flow Test Diagnostic Device.

Trypanosoma vivax is one of the causative agents of Animal African Trypanosomosis in cattle, which is endemic in sub-Saharan Africa and transmitted primarily by the bite of the tsetse fly vector. The parasite can also be mechanically transmitted, and this has allowed its spread to South America. Diagnostics are limited for this parasite and in farm settings diagnosis is mainly symptom-based. We set out to identify, using a proteomic approach, candidate diagnostic antigens to develop into an easy to use pen-side lateral flow test device. Two related members the invariant surface glycoprotein family, TvY486_0045500 and TvY486_0019690, were selected. Segments of these antigens, lacking N-terminal signal peptides and C-terminal transmembrane domains, were expressed in E. coli. Both were developed into ELISA tests and one of them, TvY486_0045500, was developed into a lateral flow test prototype. The tests were all evaluated blind with 113 randomised serum samples, taken from 37 calves before and after infection with T. vivax or T. congolense. The TvY486_0045500 and TvY486_0019690 ELISA tests gave identical sensitivity and specificity values for T. vivax infection of 94.5% (95% CI, 86.5% to 98.5%) and 88.0% (95% CI, 75.7% to 95.5%), respectively, and the TvY486_0045500 lateral flow test prototype a sensitivity and specificity of 92.0% (95% CI, 83.4% to 97.0%) and 89.8% (95% CI, 77.8% to 96.6%), respectively. These data suggest that recombinant TvY486_0045500 shows promise for the development of a pen-side lateral flow test for the diagnosis of T. vivax animal African trypanosomosis.

Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human African trypanosomiasis.

BACKGROUND: The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT. METHODOLOGY/PRINCIPLE FINDINGS: Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank. CONCLUSION/SIGNIFICANCE: In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.

Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device.

BACKGROUND: The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens. METHODOLOGY/PRINCIPAL FINDINGS: We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device. CONCLUSIONS/SIGNIFICANCE: Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

Regulation of cyclin D1 RNA stability by SNIP1.

Cyclin D1 expression represents one of the key mitogen-regulated events during the G(1) phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP-associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.

The cyclin-dependent kinase inhibitors p15INK4B and p21CIP1 are critical regulators of fibrillar collagen-induced tumor cell cycle arrest.

The extracellular matrix is a crucial component in determining cell fate. Fibrillar collagen in its native form inhibits cell proliferation, whereas in its monomeric form it stimulates proliferation. The observation of elevated levels of p27(KIP1) in cells plated in the presence of fibrillar collagen has led to the assumption that this kinase inhibitor was responsible for cell cycle arrest on fibrillar collagen. Here we provide evidence that p15(INK4b), rather than p27(KIP1), is the cyclin-dependent kinase inhibitor responsible for G0/G1 arrest of human melanoma cells grown on fibrillar collagen. Additionally, we demonstrate that fibrillar collagen can also arrest cells at the G2 phase, which is mediated in part by p21(CIP1). Our data, in addition to identifying cyclin-dependent kinase inhibitors important in cell cycle arrest mediated by fibrillar collagen, demonstrate the complexity of cell cycle regulation and indicate that modulating a single cyclin-dependent kinase inhibitor does not disrupt cell proliferation in the presence of fibrillar collagen.

Discoidin domain receptor 2 mediates tumor cell cycle arrest induced by fibrillar collagen.

During malignant invasion tumor cells establish contact with extracellular matrix proteins, including fibrillar collagen. In addition to providing a physical barrier against invasion, fibrillar collagen also restricts cell proliferation. It has been assumed that the growth regulatory activity of fibrillar collagen is the result of an indirect restrictive effect on cell spreading and cytoskeletal organization. Here we provide evidence for a direct inhibitory effect of fibrillar collagen on proliferation of human melanoma and fibrosarcoma cells that involves activation of the tyrosine kinase discoidin domain receptor 2 and is independent of effects on cell spreading. Cells plated in the presence of fibrillar collagen were growth arrested in the G0/G1 phase of the cell cycle. However treatment with the tyrosine kinase inhibitor genistein, down-regulation of discoidin domain receptor 2, or collagen deglycosylation that prevents discoidin domain receptor 2 activation allowed cells to enter the cell cycle in the presence of fibrillar collagen without a requirement for spreading and actin organization. Our data provide evidence for a novel direct mechanism by which cell contact with fibrillar collagen restricts proliferation.

Differential expression of matrix metalloproteinases during impaired wound healing of the diabetes mouse.

A number of studies have shown elevated matrix metalloproteinase expression in chronic wound fluid compared to an acute wound; however, little has been done to characterize animal models in a similar manner and thus determine their usefulness. The diabetes mouse is an animal model of type II diabetes that shows impaired dermal wound healing and has been proposed as a model of human impaired wound healing. In this study we have determined the mRNA and protein expression profiles of matrix metalloproteinases 2, 3, and 9 during the first 10 d of dermal healing for the diabetes mouse and its normally healing littermate. Additionally, human wound fluid from diabetic chronic wounds and acute surgical wounds were studied to enable a comparison of the model to the human condition. We show that during the early stages of wound healing the diabetes mouse possesses significantly reduced protein levels of pro-matrix metalloproteinases 2 and 9 within the wound tissue and active matrix metalloproteinase 3 within the fluid. Pro-matrix metalloproteinase 3 levels are also significantly reduced in the diabetes mouse during the later stages of healing. These differences may be contributing to the impaired healing of the diabetes mouse; however, they differ from the human data presented here, which show elevated matrix metalloproteinase 2 and reduced matrix metalloproteinase 9 in human diabetic chronic wound fluid compared to acute wound fluid. Therefore, although clearly showing the importance of appropriate matrix metalloproteinase regulation for normal acute wound healing to occur, the diabetes mouse may not be an ideal model for study of matrix metalloproteinase involvement in human chronic wound healing.

Polycyclic aromatic hydrocarbons in the indoor and outdoor air of three cities in the U.S.

The indoor and outdoor concentrations of 30 polycyclic aromatic hydrocarbons (PAHs) were measured in 55 nonsmoking residences in three urban areas during June 1999-May 2000. The data represent the subset of samples collected within the Relationship of Indoor, Outdoor, and Personal Air study (RIOPA). The study collected samples from homes in Los Angeles, CA, Houston, TX, and Elizabeth, NJ. In the outdoor samples, the total PAH concentrations (sigmaPAH) were 4.2-64 ng m(-3) in Los Angeles, 10-160 ng m(-3) in Houston, and 12-110 ng m(-3) in Elizabeth. In the indoor samples, the concentrations of sigmaPAH were 16-220 ng m(-3) in Los Angeles, 21-310 ng m(-3) in Houston, and 22-350 ng m(-3) in Elizabeth. The PAH profiles of low molecular weight PAHs (3-4 rings) in the outdoor samples from the three cities were not significantly different. In contrast, the profiles of 5-7-ring PAHs in thesethree citieswere significantlydifferent, which suggested different dominant PAH sources. The signatures of 5-7-ring PAHs in the indoor samples in each city were similar to the outdoor profiles, which suggested that indoor concentrations of 5-7-ring PAHs were dominated by outdoor sources. Indoor-to-outdoor ratios of the PAH concentrations showed that indoor sources had a significant effect on indoor concentrations of 3-ring PAHs and a smaller effect on 4-ring PAHs and that outdoor sources dominated the indoor concentrations of 5-7-ring PAHs.