Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

[Background]: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection.[Methods]: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines.[Results]: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells.[Conclusion]: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.Supported by the Austrian Science Fund (FWF, DK-MOLIN W1241 and the “Cluster of Excellence: Microbiomes Drive Planetary Health”) and by the Spanish Ministry of Science and Innovation under Grants (PID2021-123795OB-I00, PID2020-115506RB-I00) [Ministerio de Ciencia, Innovación y Universidades (MCIU)/Agencia Estatal de Investigación (AEI)/European Regional Development Fund (FEDER, EU)].Peer reviewe

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

BackgroundHelicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection.MethodsWe analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines.ResultsIn biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells.ConclusionH. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy

Sensitivity and Specificity of In situ Proximity Ligation for Protein Interaction Analysis in a Model of Steatohepatitis with Mallory-Denk Bodies

The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8(-/-) and Krt18(-/-) mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol

Search and seizure and its lawfulness in criminal proceedings

Bakalaura darba tēma ir “Kratīšana un tās tiesiskums kriminālprocesā”. Kratīšana ir viena no izmeklēšanas darbībām, kuras saturs ir telpas, apvidus teritorijas, transportlīdzekļa un atsevišķas personas piespiedu pārmeklēšana, nolūkā atrast un izņemt meklējamos objektus, ja ir pietiekams pamats uzskatīt, ka meklējamais objekts atrodas kratīšanas vietā. Ar kratīšanas palīdzību tiek sasniegts taisnīgs kriminālprocesa likuma noregulējums, taču šīs darbības laikā tiek aizskartas cilvēka pamattiesības, līdz ar to darba mērķis ir noskaidrot, kā var veikt kratīšanu nepārkāpjot pamattiesības, un vai tās Latvija tiek ievērotas. Darbā uzmanība pievērsta tam, kā kratīšanas definīcija atšķiras trijās valstīs (Latvijā, Krievijā, Amerikas Savienotas Valstīs (turpmāk – ASV)). Tiks apskatīta katra kratīšanas objekta būtība, ka arī izskatīts kratīšanas pamats jeb kādi lēmumi nepieciešami, lai varētu veikt kratīšanu un vai vispār tie ir vajadzīgi. Tiks noskaidrota kāda ir kratīšanas kārtība un vai tā atšķiras dažādiem kratīšanas objektiem. Kā arī darbā tiks pētīta kratīšanas rezultātu fiksēšana. Darba pamatā tiks izmantots spēka esošais Kriminālprocesa likums un citu valstu normatīvie akti, zinātnieku publikācijas, grāmatas. Darbā analizēta Latvijas un ārvalstu tiesu prakse. Pētījumā tiks izvirzīti secinājumi attiecība uz izskatīto tēmu.Bachelor’s work thesis is – search and seizure and its lawfulness in criminal proceedings. Search and seizure is one of investigative steps whose goal is the compulsory search of a room, teritorry, vehicle or a person with the goal of seizing the sought after object if there is enough evidence to suggest that this object is to be found in the searchable location. Search and seizure helps in achieving just regulation of criminal procedings, but it is imperative that during these actions no human rights must be overstepped. So the goal of this thesis is to establish how to conclude fair and just search and seizure and wheter in Latvia it is done with the human rights in mind. During this thesis apecial attention will be brought to the differences in the definition of search and seizure in three states (Latvia, Russia, USA). Also the meaning of each searchable object will be analyzed. Basis for such a search or – what rulings are needed for the conduction of a search and are they even needed. Also will be determined the procedure of search and whether it even differs with each searchable object and how the results are documented. As the base sources of information will be used Criminal law of Latvia, regulatory enactmens of other states, scientific publications, books, articles and journals. In addition will be used court cases of Latvia and other states. In the thesis there will be highlighted conclusions in relation to the researchable topic

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

BACKGROUND:Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. METHODS:Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. RESULTS:All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. CONCLUSION:PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment

Testing of ABs to keratin and p62 in mouse liver and demonstration of equivalent binding properties of ABs used in IF and <i>is</i>PLA.

<p>IF staining of normal mouse liver tissues (Swiss Albino, SWA fed a standard diet) with antibodies to K8 (green), p62 (red) (<b>A</b>) and for K18 (green) and p62 (red) (<b>B</b>). Image (<b>C</b>) shows that the two different ABs directed to K8 (rat-anti-K8 [Troma I] and mouse-anti-K8 [Ks 8.7 Progen]) used for <i>is</i>PLA and IF or DIIF recognize identical structures in liver from a DDC fed SWA mouse. Scale bars: 20 µm.</p

<i>is</i>PLA for 12 week-DDC treated mice.

<p>Images (<b>A, B</b>) show <i>is</i>PLA signal (red) using ABs to p62 and K8 (<b>A</b>) and p62 and K18 (<b>B</b>). (<b>C</b>) shows a combined visualization of p62 and K8 in <i>is</i>PLA (red) and K8 in IF (green). (<b>D</b>) shows a combined visualization of p62 and K18 in <i>is</i>PLA (red) and K18 IF (green). In images (<b>C</b>) and (<b>D</b>) note that even very small <i>is</i>PLA positive structures are visible (arrowhead), demonstrating the high sensitivity of <i>is</i>PLA. However, there were also larger MDB-like keratin aggregates that are negative for <i>is</i>PLA stain (empty arrow), suggesting the absence of p62. Scale bars: 20 µm.</p

Technical controls performed on mice with and without DDC treatment.

<p>In order to complement the biological controls (knockout mice) we performed a number of technical controls to make sure that no cross reactions between the different reagents might lead to false results.</p

Influence of channel amplification on demonstration of antigen colocalization in <i>is</i>PLA and IF in 12 week-DDC treated <i>krt18^−/−</i> mice.

<p>(<b>A, B, C</b>) Show different levels of digital amplification of the green channel (IF for K8) and constant amplification of the red channel showing <i>is</i>PLA for K8 and p62. Arrows indicate a MDB-like aggregate that was constantly positive for K8 but negative for p62. Arrowheads indicate MDBs which were positive in <i>is</i>PLA for p62 and K8 but IF showed only presence of K8 (yellow merged signal) in <i>is</i>PLA positive MDBs after maximal amplification of the green channel. Scale bars: 20 µm.</p