Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade.

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a "changing of the guards" between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.

CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke.

CD4+ T lymphocytes are key mediators of tissue damage after ischemic stroke. However, their infiltration kinetics and interactions with other immune cells in the delayed phase of ischemia remain elusive. We hypothesized that CD4+ T cells facilitate delayed autoreactive B cell responses in the brain, which have been previously linked to post-stroke cognitive impairment (PSCI). Therefore, we treated myelin oligodendrocyte glycoprotein T cell receptor transgenic 2D2 mice of both sexes with anti-CD4 antibody following 60-minute middle cerebral artery occlusion and assessed lymphocyte infiltration for up to 72 days. Anti-CD4-treatment eliminated CD4+ T cells from the circulation and ischemic brain for 28 days and inhibited B cell infiltration into the brain, particularly in animals with large infarcts. Absence of CD4+ T cells did not influence infarct maturation or survival. Once the CD4+ population recovered in the periphery, both CD4+ T and B lymphocytes entered the infarct site forming follicle-like structures. Additionally, we provide further evidence for PSCI that could be attenuated by CD4 depletion. Our findings demonstrate that CD4+ T cells are essential in delayed B cell infiltration into the ischemic brain after stroke. Importantly, lymphocyte infiltration after stroke is a long-lasting process. As CD4 depletion improved cognitive functions in an experimental set-up, these findings set the stage to elaborate more specific immune modulating therapies in treating PSCI.

Puumala Virus in Bank Voles, Lithuania.

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe.

Efficacy and safety of intratracheal IFN-γ treatment to reverse stroke-induced susceptibility to pulmonary bacterial infections.

Stroke-induced immunosuppression contributes to the development of stroke-associated pneumonia (SAP). Experiments in mice demonstrated that apoptosis of IFN-γ producing cells and reduced IFN-γ secretion resulted in impaired immune responses and the development of pneumonia after middle cerebral artery occlusion (MCAo). In the present study, we investigated the efficacy of intratracheal IFN-γ treatment to prevent SAP and demonstrated that modest benefits on pulmonary cytokine response in IFN-γ treated stroke mice did not prevent spontaneously developing infections and even slightly reduced bacterial clearance of aspirated pneumococci. Our results suggest that pulmonary IFN-γ treatment is not an effective preventive measure for SAP.

Impact of Key Nicotinic AChR Subunits on Post-Stroke Pneumococcal Pneumonia.

Pneumonia is the most frequent severe medical complication after stroke. An overactivation of the cholinergic signaling after stroke contributes to immunosuppression and the development of spontaneous pneumonia caused by Gram-negative pathogens. The α7 nicotinic acetylcholine receptor (α7nAChR) has already been identified as an important mediator of the anti-inflammatory pathway after stroke. However, whether the α2, α5 and α9/10 nAChR expressed in the lung also play a role in suppression of pulmonary innate immunity after stroke is unknown. In the present study, we investigate the impact of various nAChRs on aspiration-induced pneumonia after stroke. Therefore, α2, α5, α7 and α9/10 nAChR knockout (KO) mice and wild type (WT) littermates were infected with Streptococcus pneumoniae (S. pneumoniae) three days after middle cerebral artery occlusion (MCAo). One day after infection pathogen clearance, cellularity in lung and spleen, cytokine secretion in bronchoalveolar lavage (BAL) and alveolar-capillary barrier were investigated. Here, we found that deficiency of various nAChRs does not contribute to an enhanced clearance of a Gram-positive pathogen causing post-stroke pneumonia in mice. In conclusion, these findings suggest that a single nAChR is not sufficient to mediate the impaired pulmonary defense against S. pneumoniae after experimental stroke.

Spatial and Temporal Evolutionary Patterns in Puumala Orthohantavirus (PUUV) S Segment.

The S segment of bank vole (Clethrionomys glareolus)-associated Puumala orthohantavirus (PUUV) contains two overlapping open reading frames coding for the nucleocapsid (N) and a non-structural (NSs) protein. To identify the influence of bank vole population dynamics on PUUV S segment sequence evolution and test for spillover infections in sympatric rodent species, during 2010-2014, 883 bank voles, 357 yellow-necked mice (Apodemus flavicollis), 62 wood mice (A. sylvaticus), 149 common voles (Microtus arvalis) and 8 field voles (M. agrestis) were collected in Baden-Wuerttemberg and North Rhine-Westphalia, Germany. In total, 27.9% and 22.3% of bank voles were positive for PUUV-reactive antibodies and PUUV-specific RNA, respectively. One of eight field voles was PUUV RNA-positive, indicating a spillover infection, but none of the other species showed evidence of PUUV infection. Phylogenetic and isolation-by-distance analyses demonstrated a spatial clustering of PUUV S segment sequences. In the hantavirus outbreak years 2010 and 2012, PUUV RNA prevalence was higher in our study regions compared to non-outbreak years 2011, 2013 and 2014. NSs amino acid and nucleotide sequence types showed temporal and/or local variation, whereas the N protein was highly conserved in the NSs overlapping region and, to a lower rate, in the N alone coding part.

Loss of Mismatch-repair Protein Expression and Microsatellite Instability in Upper Tract Urothelial Carcinoma and Clinicopathologic Implications.

BACKGROUND: Upper tract urothelial carcinoma (UTUC) may arise in the setting of hereditary non-polyposis colorectal cancer (Lynch syndrome [LS]) or sporadically. Variable frequencies of microsatellite instability (MSI) were found in UTUC. For advanced solid MSI tumors, targeted therapy with programmed death-ligand 1 inhibitors is available. Therefore, we aimed to determine the prevalence of mismatch repair (MMR) protein loss and MSI in UTUC using a tissue microarray approach and further molecular and correlation analysis. MATERIALS AND METHODS: We studied the immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2 on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 128 patients with UTUC. MSI analysis was performed in 79 cases with deficient MMR protein expression, and/or in patients aged 60 years and below, and/or other tumors possibly related to LS. RESULTS: Loss of MMR protein expression was seen in 24 (18.8%) of 128 cases. MSI analysis revealed MSI-high in 29, MSI-low in 7 cases. The Fisher exact test demonstrated significant differences between MSI and loss of MMR protein expression, clinically possible LS, tumor growth pattern, inverted growth pattern, and death (P < .001, P < .001, P = .002, P = .003, and P = .033, respectively). MSI does not appear to influence survival (overall and progression-free), but there was a significant shorter progression-free survival in MSI-high versus MSS patients who had received chemotherapy. CONCLUSION: The frequency of MSI in UTUC was 36 (28.1%) of 128 patients with a good accuracy of immunohistochemistry. In daily practice, MSI screening especially is recommended in patients with advanced UTUC and inverted papillary tumor growth pattern with the aim of screening patients for possible targeted therapy.