Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma.

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.

Neoadjuvant immunotherapy in gastrointestinal cancers - The new standard of care?

The development of immune checkpoint inhibitors (ICI) offers novel treatment possibilities for solid cancers, with the crucial benefit of providing higher cure rates. These agents have become part of standard treatments in the metastatic and adjuvant setting for select cancers, such as melanoma, non-small cell lung cancer (NSCLC) or urological malignancies. Currently, there is ample clinical interest in employing ICI in a neoadjuvant setting with a curative intent. This approach is especially supported by the scientific rationale that ICI primarily stimulate the host immune system to eradicate tumor cells, rather than being inherently cytotoxic. Aside from tumor downstaging, neoadjuvant immunotherapy offers the potential of an in situ cancer vaccination, leading to a systemic adjuvant immunological effect after tumor resection. Moreover, preclinical data clearly demonstrate a synergistic effect of ICI with radiotherapy (RT), chemoradiotherapy (CRT) or chemotherapy (ChT). This review harmonizes preclinical concepts with real world data (RWD) in the field of neoadjuvant ICI in gastrointestinal (GI) cancers and discusses their limitations. We believe this is a crucial approach, since up to now, neoadjuvant strategies have been primarily developed by clinicians, whereas the advances in immunotherapy primarily originate from preclinical research. Currently there is limited published data on neoadjuvant ICI in GI cancers, even though neoadjuvant treatments including RT, CRT or ChT are frequently employed in locally advanced/oligometastatic GI cancers (i.e. rectal, pancreatic, esophagus, stomach, etc.). Utilizing established therapies in combination with ICI provides an abundance of opportunities for innovative treatment regimens to further improve survival rates.

Evaluation of Intestinal Fibrosis with 68Ga-FAPI PET/MR Enterography in Crohn Disease.

Background In Crohn disease, differentiation between active intestinal inflammation and fibrosis has implications for treatment, but current imaging modalities are not reliably accurate. Purpose To evaluate the predictive value of gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor (FAPI) PET/MR enterography for the assessment of bowel wall fibrosis in Crohn disease. Materials and Methods In this prospective single-center study, consecutive participants with Crohn disease and obstructive symptoms underwent preoperative 68Ga-FAPI PET/MR enterography from May 2021 to January 2022. Histopathologic analysis of resected bowel segments was performed to grade active inflammation (A0-A2) and fibrosis (F0-F2), which served as the reference standard. The fibroblast activation protein (FAP) expression in bowel wall layers was analyzed immunohistochemically for each layer. 68Ga-FAPI-derived maximum standardized uptake value (SUVmax) was compared with histopathologic results by using mixed-model analysis of variance and Bonferroni-corrected post hoc tests. Results In 14 participants (mean age, 45 years ± 9 [SD]; 10 men), fibrosis was diagnosed histopathologically in 28 of 51 bowel segments (grade F1, n = 14; grade F2, n = 14). Mean SUVmax was higher in segments with fibrosis than without (7.6 vs 2.0; P < .001). In severe fibrosis, mean SUVmax was higher than in mild to moderate fibrosis (8.9 ± 0.9 vs 6.2 ± 0.9; P = .045). Bowel segments with isolated active inflammation had lower mean 68Ga-FAPI uptake than segments with combined active inflammation and fibrosis (SUVmax, 3.2 ± 0.4 vs 8.1 ± 0.1; P = .005). With an SUVmax cutoff value of 3.5, the area under the receiver operating characteristic curve for the prediction of fibrosis was 0.94 (95% CI: 0.9, 1.0), with sensitivity of 26 of 28 segments (93%) and specificity of five of six segments (83%). 68Ga-FAPI-derived SUVmax correlated with FAP expression across all bowel layers (R2 = 0.50, P < .001). Conclusion Higher gallium 68 fibroblast activation protein inhibitor uptake at PET/MR enterography was associated with histopathologically assessed bowel wall fibrosis in participants with Crohn disease, suggesting diagnostic potential for treatment decisions. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by O'Shea in this issue.

Selective Eradication of Colon Cancer Cells Harboring PI3K and/or MAPK Pathway Mutations in 3D Culture by Combined PI3K/AKT/mTOR Pathway and MEK Inhibition.

Colorectal cancer (CRC) is the second deadliest cancer in the world. Besides APC and p53 alterations, the PI3K/AKT/MTOR and MAPK pathway are most commonly mutated in CRC. So far, no treatment options targeting these pathways are available in routine clinics for CRC patients. We systematically analyzed the response of CRC cells to the combination of small molecular inhibitors targeting the PI3K and MAPK pathways. We used CRC cells in 2D, 3D spheroid, collagen gel cultures and freshly isolated organoids for drug response studies. Readout for drug response was spheroid or organoid growth, spheroid outgrowth, metabolic activity, Western blotting and immunofluorescence. We found profound tumor cell destruction under treatment with a combination of Torin 1 (inhibiting mTOR), MK2206 (targeting AKT) and selumetinib (inhibiting MEK) in 3D but not in 2D. Induction of cell death was due to apoptosis. Western blot analysis revealed efficient drug action. Gedatolisib, a dual PI3K/mTOR inhibitor, could replace Torin1/MK2206 with similar efficiency. The presence of PI3K and/or RAS-RAF-MAPK pathway mutations accounted for treatment responsiveness. Here, we identified a novel, efficient therapy, which induced proliferation stop and tumor cell destruction in vitro based on the genetic background. These preclinical findings show promise to further test this combi-treatment in vivo in mice and to potentially develop a mutation specific targeted therapy for CRC patients.

Globe-sparing surgical treatment for periocular malignancies with anterior orbital invasion: a consecutive case series.

PURPOSE: Orbital exenteration of periocular tumors complicated by orbital invasion is a heavy burden for patients and leads to disfiguring cosmesis and loss of vision. Here, we report our experience with globe-sparing surgery in a series of patients with periocular malignancies other than basal cell carcinoma (BCC), all exhibiting anterior orbital invasion. METHODS: In this consecutive case series, we examined medical records of all patients between 2000 and 2018 with periocular malignancies (other than BCC) invading the anterior orbit (without extraocular muscle or scleral invasion) treated by one orbital surgeon (DP). The main outcome measures included local recurrence, regional and distant metastasis, survival, and visual acuity. RESULTS: Nine patients were identified. Of the non-BCC cancers invading the orbit, squamous cell carcinoma (SCC) (44.4%) was the most prevalent type in our series. Excision included the removal of visibly distinguishable tumor and a free clinical margin of up to 5 mm with histological confirmation of radicality of the invasive tumor component. Reconstruction was achieved by a variety of oculoplastic reconstructive procedures. At a mean follow-up of 70 months (range 11-177 months), 8 out of 9 patients were still alive. Recurrence occurred in two patients with conjunctival melanoma (CM), and they were again treated with wide excision. Postoperative visual acuity remained stable or improved. CONCLUSION: This retrospective case series demonstrates that globe-sparing excisional surgery can be considered in selected cases of periocular malignancies other than BCC with anterior orbital invasion, thus avoiding cosmetic disfigurement and loss of vision due to orbital exenteration.

Early Postoperative Basal Insulin Therapy versus Standard of Care for the Prevention of Diabetes Mellitus after Kidney Transplantation: A Multicenter Randomized Trial.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) might be preventable. METHODS: This open-label, multicenter randomized trial compared 133 kidney transplant recipients given intermediate-acting insulin isophane for postoperative afternoon glucose ≥140 mg/dl with 130 patients given short-acting insulin for fasting glucose ≥200 mg/dl (control). The primary end point was PTDM (antidiabetic treatment or oral glucose tolerance test-derived 2 hour glucose ≥200 mg/dl) at month 12 post-transplant. RESULTS: In the intention-to-treat population, PTDM rates at 12 months were 12.2% and 14.7% in treatment versus control groups, respectively (odds ratio [OR], 0.82; 95% confidence interval [95% CI], 0.39 to 1.76) and 13.4% versus 17.4%, respectively, at 24 months (OR, 0.71; 95% CI, 0.34 to 1.49). In the per-protocol population, treatment resulted in reduced odds for PTDM at 12 months (OR, 0.40; 95% CI, 0.16 to 1.01) and 24 months (OR, 0.54; 95% CI, 0.24 to 1.20). After adjustment for polycystic kidney disease, per-protocol ORs for PTDM (treatment versus controls) were 0.21 (95% CI, 0.07 to 0.62) at 12 months and 0.35 (95% CI, 0.14 to 0.87) at 24 months. Significantly more hypoglycemic events (mostly asymptomatic or mildly symptomatic) occurred in the treatment group versus the control group. Within the treatment group, nonadherence to the insulin initiation protocol was associated with significantly higher odds for PTDM at months 12 and 24. CONCLUSIONS: At low overt PTDM incidence, the primary end point in the intention-to-treat population did not differ significantly between treatment and control groups. In the per-protocol analysis, early basal insulin therapy resulted in significantly higher hypoglycemia rates but reduced odds for overt PTDM-a significant reduction after adjustment for baseline differences-suggesting the intervention merits further study.Clinical Trial registration number: NCT03507829.

The Role of Telocytes and Telocyte-Derived Exosomes in the Development of Thoracic Aortic Aneurysm.

A hallmark of thoracic aortic aneurysms (TAA) is the degenerative remodeling of aortic wall, which leads to progressive aortic dilatation and resulting in an increased risk for aortic dissection or rupture. Telocytes (TCs), a distinct type of interstitial cells described in many tissues and organs, were recently observed in the aortic wall, and studies showed the potential regulation of smooth muscle cell (SMC) homeostasis by TC-released shed vesicles. The purpose of the present work was to study the functions of TCs in medial degeneration of TAA. During aneurysmal formation an increase of aortic TCs was identified in human surgical specimens of TAA-patients, compared to healthy thoracic aortic (HTA)-tissue. We found the presence of epithelial progenitor cells in the adventitial layer, which showed increased infiltration in TAA samples. For functional analysis, HTA- and TAA-telocytes were isolated, characterized, and compared by their protein levels, mRNA- and miRNA-expression profiles. We detected TC and TC-released exosomes near SMCs. TAA-TC-exosomes showed a significant increase of the SMC-related dedifferentiation markers KLF-4-, VEGF-A-, and PDGF-A-protein levels, as well as miRNA-expression levels of miR-146a, miR-221 and miR-222. SMCs treated with TAA-TC-exosomes developed a dedifferentiation-phenotype. In conclusion, the study shows for the first time that TCs are involved in development of TAA and could play a crucial role in SMC phenotype switching by release of extracellular vesicles.

Impacts of Telomeric Length, Chronic Hypoxia, Senescence, and Senescence-Associated Secretory Phenotype on the Development of Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) is an age-related and life-threatening vascular disease. Telomere shortening is a predictor of age-related diseases, and its progression is associated with premature vascular disease. The aim of the present work was to investigate the impacts of chronic hypoxia and telomeric DNA damage on cellular homeostasis and vascular degeneration of TAA. We analyzed healthy and aortic aneurysm specimens (215 samples) for telomere length (TL), chronic DNA damage, and resulting changes in cellular homeostasis, focusing on senescence and apoptosis. Compared with healthy thoracic aorta (HTA), patients with tricuspid aortic valve (TAV) showed telomere shortening with increasing TAA size, in contrast to genetically predisposed bicuspid aortic valve (BAV). In addition, TL was associated with chronic hypoxia and telomeric DNA damage and with the induction of senescence-associated secretory phenotype (SASP). TAA-TAV specimens showed a significant difference in SASP-marker expression of IL-6, NF-κB, mTOR, and cell-cycle regulators (γH2AX, Rb, p53, p21), compared to HTA and TAA-BAV. Furthermore, we observed an increase in CD163+ macrophages and a correlation between hypoxic DNA damage and the number of aortic telocytes. We conclude that chronic hypoxia is associated with telomeric DNA damage and the induction of SASP in a diseased aortic wall, promising a new therapeutic target.

Intensive care unit occupancy predictions in the COVID-19 pandemic based on age-structured modelling and differential flatness.

The COVID-19 pandemic confronts governments and their health systems with great challenges for disease management. In many countries, hospitalization and in particular ICU occupancy is the primary measure for policy makers to decide on possible non-pharmaceutical interventions. In this paper a combined methodology for the prediction of COVID-19 case numbers, case-specific hospitalization and ICU admission rates as well as hospital and ICU occupancies is proposed. To this end, we employ differential flatness to provide estimates of the states of an epidemiological compartmental model and estimates of the unknown exogenous inputs driving its nonlinear dynamics. A main advantage of this method is that it requires the reported infection cases as the only data source. As vaccination rates and case-specific ICU rates are both strongly age-dependent, specifically an age-structured compartmental model is proposed to estimate and predict the spread of the epidemic across different age groups. By utilizing these predictions, case-specific hospitalization and case-specific ICU rates are subsequently estimated using deconvolution techniques. In an analysis of various countries we demonstrate how the methodology is able to produce real-time state estimates and hospital/ICU occupancy predictions for several weeks thus providing a sound basis for policy makers.

Telocytes in the human ascending aorta: Characterization and exosome-related KLF-4/VEGF-A expression.

Telocytes (TCs), a novel interstitial cell entity promoting tissue regeneration, have been described in various tissues. Their role in inter-cellular signalling and tissue remodelling has been reported in almost all human tissues. This study hypothesizes that TC also contributes to tissue remodelling and regeneration of the human thoracic aorta (HTA). The understanding of tissue homeostasis and regenerative potential of the HTA is of high clinical interest as it plays a crucial role in pathogenesis from aortic dilatation to lethal dissection. Therefore, we obtained twenty-five aortic specimens of heart donors during transplantation. The presence of TCs was detected in different layers of aortic tissue and characterized by immunofluorescence and transmission electron microscopy. Further, we cultivated and isolated TCs in highly differentiated form identified by positive staining for CD34 and c-kit. Aortic-derived TC was characterized by the expression of PDGFR-α, PDGFR-ß, CD29/integrin ß-1 and αSMA and the stem cell markers Nanog and KLF-4. Moreover, TC exosomes were isolated and characterized for soluble angiogenic factors by Western blot. CD34+ /c-kit+ TCs shed exosomes containing the soluble factors VEGF-A, KLF-4 and PDGF-A. In summary, TC occurs in the aortic wall. Correspondingly, exosomes, derived from aortic TCs, contain vasculogenesis-relevant proteins. Understanding the regulation of TC-mediated aortic remodelling may be a crucial step towards designing strategies to promote aortic repair and prevent adverse remodelling.

Estimation of exogenous drivers to predict COVID-19 pandemic using a method from nonlinear control theory.

The currently ongoing COVID-19 pandemic confronts governments and their health systems with great challenges for disease management. Epidemiological models play a crucial role, thereby assisting policymakers to predict the future course of infections and hospitalizations. One difficulty with current models is the existence of exogenous and unmeasurable variables and their significant effect on the infection dynamics. In this paper, we show how a method from nonlinear control theory can complement common compartmental epidemiological models. As a result, one can estimate and predict these exogenous variables requiring the reported infection cases as the only data source. The method allows to investigate how the estimates of exogenous variables are influenced by non-pharmaceutical interventions and how imminent epidemic waves could already be predicted at an early stage. In this way, the concept can serve as an "epidemometer" and guide the optimal timing of interventions. Analyses of the COVID-19 epidemic in various countries demonstrate the feasibility and potential of the proposed approach. The generic character of the method allows for straightforward extension to different epidemiological models.

Plasma metabolites associated with colorectal cancer stage: Findings from an international consortium.

Colorectal cancer is the second most common cause of cancer-related death globally, with marked differences in prognosis by disease stage at diagnosis. We studied circulating metabolites in relation to disease stage to improve the understanding of metabolic pathways related to colorectal cancer progression. We investigated plasma concentrations of 130 metabolites among 744 Stages I-IV colorectal cancer patients from ongoing cohort studies. Plasma samples, collected at diagnosis, were analyzed with liquid chromatography-mass spectrometry using the Biocrates AbsoluteIDQ™ p180 kit. We assessed associations between metabolite concentrations and stage using multinomial and multivariable logistic regression models. Analyses were adjusted for potential confounders as well as multiple testing using false discovery rate (FDR) correction. Patients presented with 23, 28, 39 and 10% of Stages I-IV disease, respectively. Concentrations of sphingomyelin C26:0 were lower in Stage III patients compared to Stage I patients (pFDR < 0.05). Concentrations of sphingomyelin C18:0 and phosphatidylcholine (diacyl) C32:0 were statistically significantly higher, while citrulline, histidine, phosphatidylcholine (diacyl) C34:4, phosphatidylcholine (acyl-alkyl) C40:1 and lysophosphatidylcholines (acyl) C16:0 and C17:0 concentrations were lower in Stage IV compared to Stage I patients (pFDR < 0.05). Our results suggest that metabolic pathways involving among others citrulline and histidine, implicated previously in colorectal cancer development, may also be linked to colorectal cancer progression.

Crohn's disease: prevalence, MR features, and clinical significance of enteric and colonic sinus tracts.

OBJECTIVES: Enteric and colonic sinus tracts are inflammatory complications that precede intestinal fistulas in patients with Crohn's disease (CD). The aim of this study was to retrospectively determine the prevalence, morphologic features, and outcome of sinus tracts using MR imaging. METHODS: A consecutive cohort of 642 patients with known CD, referred for MR enterography or MR enteroclysis (study period 01/2014-09/2019), was evaluated retrospectively for the presence of sinus tracts, their locations, presence and length of coexisting strictures, bowel wall thickness, CDMI score, upstream dilation, and bowel distension. Clinical outcome was assessed using medical records. For metric data, means and standard deviation, as well as one-way ANOVA and Pearson's correlation coefficient, were calculated. RESULTS: In 36/642 patients with CD undergoing MRE, 49 sinus tracts (forty in small intestine, nine in left-sided colon) were detected with a prevalence of 6.9% in patients with MR-visible signs of CD (n = 519, overall prevalence of 5.6%). Mean segmental bowel wall thickness was 8.9 mm, and mean CDMI score was 9.3. All sinus tracts were located within a stenotic segment, showing mesenteric orientation within the small bowel and upstream dilation in 13 patients. Of 36 patients, 19 underwent immediate surgery and seven developed clinical progression within the segment containing the sinus tract. CONCLUSIONS: Sinus tracts occur in 6.9% of patients with visible signs of CD. They are located within stenotic, severely thickened bowel segments with high MR inflammation scores. Their detection is clinically important, because they indicate a more aggressive phenotype and, if left untreated, may show severe progression. KEY POINTS: • Sinus tracts occur in 6.9% of patients with MR-visible signs of Crohn's disease. • Sinus tracts are a radiological indicator of early penetrating Crohn's disease, with a high risk of progression, and require dedicated treatment. • Sinus tracts can be recognized by characteristic findings and typically occur in stenotic, severely thickened bowel segments with high MR inflammation scores.

Direct EPR detection of atomic nitrogen in an atmospheric nitrogen plasma jet.

In this study, an atmospheric nitrogen plasma jet generated by a custom-built micro-plasma device was analyzed at room temperature by continuous wave and pulse EPR spectroscopy in real time. Transiently formed nitrogen atoms were detected without the necessity to use spin-traps or other reagents for their stabilization. In contrast to results from optical emission spectroscopy, only signals from the 4S ground state of 14N and 15N could be detected. EPR data analysis revealed an isotropic g value of 1.9971 and isotropic hyperfine coupling constants of a(14N) = (10.47 ± 0.02) MHz and a(15N) = (14.69 ± 0.02) MHz. Moreover, lifetime and relaxation data could be determined; both are discussed in terms of spectral widths and actual concentrations of the transiently formed nitrogen species within the plasma jet. The data show that the lifetimes of atomic nitrogen and charged particles such as N+ must be different, and for the latter below the observation time window of EPR spectroscopy. We demonstrate that the real-time (pulsed) EPR technique is a fast and reliable alternative to detect atomic nitrogen in atmospheric pressure plasma jets. The method may be used for a continuous monitoring of the quality of plasma jets.

Plasma metabolites associated with colorectal cancer: A discovery-replication strategy.

Colorectal cancer is known to arise from multiple tumorigenic pathways; however, the underlying mechanisms remain not completely understood. Metabolomics is becoming an increasingly popular tool in assessing biological processes. Previous metabolomics research focusing on colorectal cancer is limited by sample size and did not replicate findings in independent study populations to verify robustness of reported findings. Here, we performed a ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) screening on EDTA plasma from 268 colorectal cancer patients and 353 controls using independent discovery and replication sets from two European cohorts (ColoCare Study: n = 180 patients/n = 153 controls; the Colorectal Cancer Study of Austria (CORSA) n = 88 patients/n = 200 controls), aiming to identify circulating plasma metabolites associated with colorectal cancer and to improve knowledge regarding colorectal cancer etiology. Multiple logistic regression models were used to test the association between disease state and metabolic features. Statistically significant associated features in the discovery set were taken forward and tested in the replication set to assure robustness of our findings. All models were adjusted for sex, age, BMI and smoking status and corrected for multiple testing using False Discovery Rate. Demographic and clinical data were abstracted from questionnaires and medical records.

Circulating Free Methylated Tumor DNA Markers for Sensitive Assessment of Tumor Burden and Early Response Monitoring in Patients Receiving Systemic Chemotherapy for Colorectal Cancer Liver Metastasis.

BACKGROUND: Neoadjuvant chemotherapy (neoCTx) followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CLM). Treatment response is generally assessed using radiologic imaging after several cycles of chemotherapy. However, earlier assessment of response would be desirable since nonresponders could be switched early to an alternative chemotherapy regimen. Recent evidence suggests that circulating free methylated tumor DNA is a highly sensitive biomarker and may more accurately reflect tumor burden and treatment response than conventional markers for CRC. PATIENTS AND METHODS: Thirty-four patients with CLM who received neoCTx prior to intended hepatic resection were included in this prospective nonrandomized study. Peripheral blood plasma was collected at baseline and before each cycle of neoCTx and was then analyzed for aberrant methylation of 48 CRC-associated genes. Methylation marker levels were correlated with baseline tumor volume and treatment response and compared with the standard tumor markers CEA and CA 19-9. RESULTS: The methylation markers SEPT9, DCC, BOLL, and SFRP2 were present in all patients at baseline and displayed a stronger correlation with tumor volume than CEA and CA 19-9. Serial measurement of these methylation markers allowed for discrimination between operated and nonoperated patients already after 1 cycle of neoCTx with high sensitivity and specificity. The early dynamic changes of SEPT9 and DCC also seemed to correlate with pathohistological response. CONCLUSION: Our data suggest that serial measurements of CRC-associated methylation markers could be a particularly valuable tool for early response assessment in patients receiving neoCTx for CLM.

Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host metabolism. The complexity of virus-host interaction networks is progressively fathomed by novel metagenomic approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treatment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative abundance of encoded protein functions was associated with the ecologic niches probed. Together, these observations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.-Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T., Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

In Situ Characterization of Tissue-Resident Immune Cells by MALDI Mass Spectrometry Imaging.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/z values have the same spatial distribution as CD3+ T lymphocytes in the lymphoid follicular structures or as CD206+ macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M1 and anti-inflammatory M2 phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/z values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

Telomere Biology and Thoracic Aortic Aneurysm.

Ascending aortic aneurysms are mostly asymptomatic and present a great risk of aortic dissection or perforation. Consequently, ascending aortic aneurysms are a source of lethality with increased age. Biological aging results in progressive attrition of telomeres, which are the repetitive DNA sequences at the end of chromosomes. These telomeres play an important role in protection of genomic DNA from end-to-end fusions. Telomere maintenance and telomere attrition-associated senescence of endothelial and smooth muscle cells have been indicated to be part of the pathogenesis of degenerative vascular diseases. This systematic review provides an overview of telomeres, telomere-associated proteins and telomerase to the formation and progression of aneurysms of the thoracic ascending aorta. A better understanding of telomere regulation in the vascular pathology might provide new therapeutic approaches. Measurements of telomere length and telomerase activity could be potential prognostic biomarkers for increased risk of death in elderly patients suffering from an aortic aneurysm.

Tiotropium Respimat inhaler and the risk of death in COPD.

BACKGROUND: Tiotropium delivered at a dose of 5 µg with the Respimat inhaler showed efficacy similar to that of 18 µg of tiotropium delivered with the HandiHaler inhalation device in placebo-controlled trials involving patients with chronic obstructive pulmonary disease (COPD). Although tiotropium HandiHaler was associated with reduced mortality, as compared with placebo, more deaths were reported with tiotropium Respimat than with placebo. METHODS: In this randomized, double-blind, parallel-group trial involving 17,135 patients with COPD, we evaluated the safety and efficacy of tiotropium Respimat at a once-daily dose of 2.5 µg or 5 µg, as compared with tiotropium HandiHaler at a once-daily dose of 18 µg. Primary end points were the risk of death (noninferiority study, Respimat at a dose of 5 µg or 2.5 µg vs. HandiHaler) and the risk of the first COPD exacerbation (superiority study, Respimat at a dose of 5 µg vs. HandiHaler). We also assessed cardiovascular safety, including safety in patients with stable cardiac disease. RESULTS: During a mean follow-up of 2.3 years, Respimat was noninferior to HandiHaler with respect to the risk of death (Respimat at a dose of 5 µg vs. HandiHaler: hazard ratio, 0.96; 95% confidence interval [CI], 0.84 to 1.09; Respimat at a dose of 2.5 µg vs. HandiHaler: hazard ratio, 1.00; 95% CI, 0.87 to 1.14) and not superior to HandiHaler with respect to the risk of the first exacerbation (Respimat at a dose of 5 µg vs. HandiHaler: hazard ratio, 0.98; 95% CI, 0.93 to 1.03). Causes of death and incidences of major cardiovascular adverse events were similar in the three groups. CONCLUSIONS: Tiotropium Respimat at a dose of 5 µg or 2.5 µg had a safety profile and exacerbation efficacy similar to those of tiotropium HandiHaler at a dose of 18 µg in patients with COPD. (Funded by Boehringer Ingelheim; TIOSPIR ClinicalTrials.gov number, NCT01126437.).