| 1. |
- Gustafsson, Karin, et al.
(författare)
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Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells
- 2011
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Ingår i: BMC Immunology. - : Springer Science and Business Media LLC. - 1471-2172. ; 12, s. 3-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Shb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and develeopment. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function. Results: Shb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ TH cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production level of naïve TH cells. This suggests a TH2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern.Conclusion: Our results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.
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| 2. |
- Hjelm, Barbara, et al.
(författare)
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Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
- 2011
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 20:11, s. 1824-1835
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Tidskriftsartikel (refereegranskat)abstract
- A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.
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| 3. |
- Hjelm, Barbara, et al.
(författare)
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High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer
- 2011
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Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 5:11-12, s. 624-635
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. Experimental design: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polydonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polydonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polydonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n = 270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n = 305). RBM3-expression was detected, with high correlation between both antibodies (R = 0.81, p < 0.001). Results: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. Conclusion and clinical relevance: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.
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| 4. |
- Molin, Ylva, et al.
(författare)
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Migratory birds, ticks and Bartonella
- 2011
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Ingår i: Infection Ecology & Epidemiology. - : Informa UK Limited. - 2000-8686. ; 1, s. 5997-
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Tidskriftsartikel (refereegranskat)abstract
- Bartonella spp. infections are considered to be vector-borne zoonoses; ticks are suspected vectors of bartonellae. Migratory birds can disperse ticks infected with zoonotic pathogens such as Rickettsia and tick-borne encephalitis virus and possibly also Bartonella. Thus, in the present study 386 tick specimens collected in spring 2009 from migratory birds on the Mediterranean islands Capri and Antikythera were screened for Bartonella spp. RNA. One or more ticks were found on 2.7% of the birds. Most ticks were Hyalomma rufipes nymphs and larvae with mean infestation rates of 1.7 nymphs and 0.6 larvae per infested bird. Bartonella spp. RNA was not detected in any of the tick specimens.
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| 5. |
- Zieba, Agata, et al.
(författare)
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Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
- 2010
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 56:1, s. 99-110
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. METHODS: We used horseradish peroxidase (HRP)conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. RESULTS: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both ill cultured cells and in tissue samples. CONCLUSIONS: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be Counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.
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