Laser-heated needle for biopsy tract ablation: In vivo study of rabbit liver biopsy

PurposeTo investigate the efficacy of a newly-developed laser-heated core biopsy needle in the thermal ablation of biopsy tract to reduce hemorrhage after biopsy using in vivo rabbit’s liver model.Materials and methodsFive male New Zealand White rabbits weighed between 1.5 and 4.0 kg were anesthetized and their livers were exposed. 18 liver biopsies were performed under control group (without tract ablation, n = 9) and study group (with tract ablation, n = 9) settings. The needle insertion depth (~3 cm) and rate of retraction (~3 mm/s) were fixed in all the experiments. For tract ablation, three different needle temperatures (100, 120 and 150 °C) were compared. The blood loss at each biopsy site was measured by weighing the gauze pads before and after blood absorption. The rabbits were euthanized immediately and the liver specimens were stained with hematoxylin-eosin (H&E) for further histopathological examination (HPE).ResultsThe average blood loss in the study group was reduced significantly (p < 0.05) compared to the control group. The highest percentage of bleeding reduction was observed at the needle temperature of 150 °C (93.8%), followed by 120 °C (85.8%) and 100 °C (84.2%). The HPE results show that the laser-heated core biopsy needle was able to cause lateral coagulative necrosis up to 14 mm diameter along the ablation tract.ConclusionThe laser-heated core biopsy needle reduced hemorrhage up to 93.8% and induced homogenous coagulative necrosis along the ablation tract in the rabbits’ livers. This could potentially reduce the risk of tumor seeding in clinical settings.     

Role of oncoproteomics in the personalized management of cancer

Oncoproteomics is the term used to describe the application of proteomic technologies in oncology and parallels the related field of oncogenomics. It is now contributing to the development of personalized management of cancer. Proteomic technologies are used for the identification of biomarkers in cancer, which will facilitate the integration of diagnosis and therapy of cancer. Molecular diagnostics, laser capture microdissection and protein biochips are among the technologies that are having an important impact on oncoproteomics. The discovery of protein patterns developed by the US Food and Drug Administration/National Cancer Institute Clinical Proteomics Program is capable of distinguishing cancer and disease-free states with high sensitivity and specificity and will also facilitate the development of personalized therapy of cancer. Examples of application are given for breast and prostate cancer and a selection of companies and their collaborations that are developing application of proteomics to personalized treatment of cancer are discussed. Continued refinement of techniques and methods to determine the abundance and status of proteins in vivo holds great promise for the future study of normal cells and the pathology of associated neoplasms. Personalized cancer therapy is expected to be in the clinic by the end of the first decade of the 21st century.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).

Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2⁺ and HER2^- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2^- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2⁺ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2^- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.     

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Background

Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods

Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G⁺ neutrophils and MHC II⁺ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results

Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions

Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.     

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O₂ pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (>80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO₂, Al₂O₃, WO₃ and Ag₄O₄.     

Surface-bound basement membrane components accelerate amyloid-β peptide nucleation in air-free wells: An in vitro model of cerebral amyloid angiopathy

Cerebral amyloid angiopathy is caused by deposition of the amyloid β-peptide which consists of mainly 39–40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid β-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid β-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1 rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5 μM amyloid β(1–40) at 37 °C without air, where amyloid β(1–40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid β-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy.     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.