Myocardial Recovery, Metabolism, and Structure after Cardiac Arrest with Cardioplexol.

OBJECTIVES: Clinical studies indicate encouraging cardioprotective potential for Cardioplexol. Its cardioprotective capacities during 45 minutes of ischemia compared with pure no-flow ischemia or during 90 minutes of ischemia compared with Calafiore cardioplegia were investigated experimentally. METHODS: Forty-four rat hearts were isolated and inserted into a blood-perfused pressure-controlled Langendorff apparatus. In a first step, cardiac arrest was induced by Cardioplexol or pure no-flow ischemia lasting 45 minutes. In a second step, cardiac arrest was induced by Cardioplexol or Calafiore cardioplegia lasting 90 minutes. For both experimental steps, cardiac function, metabolic parameters, and troponin I levels were evaluated during 90 minutes of reperfusion. At the end of reperfusion, hearts were fixed, and ultrastructural integrity was examined by electron microscopy. RESULTS: Step 1: after 90 minutes of reperfusion, hearts exposed to Cardioplexol had significantly higher left ventricular developed pressure (CP-45': 74%BL vs. no-flow-45': 45%BL; p = 0.046) and significantly better maximal left ventricular relaxation (CP-45': 84%BL vs. no-flow-45': 51%BL; p = 0.012). Oxygen consumption, lactate production, and troponin levels were similar in both groups. Step 2: left ventricular developed pressure was lower (22 vs. 48% of BL; p = 0.001) and coronary flow was lower (24 vs. 53% of BL; p = 0.002) when Cardioplexol was used compared with Calafiore cardioplegia. Troponin I levels were significantly higher under Cardioplexol (358.9 vs. 106.1 ng/mL; p = 0.016). CONCLUSION: Cardioplexol significantly improves functional recovery after 45 minutes of ischemia compared with pure ischemia. However, Cardioplexol protects the myocardium from ischemia/reperfusion-related damage after 90 minutes of ischemia worse than Calafiore cardioplegia.

CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung.

The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68-79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.

SOCE-inhibitor reduced human sperm-induced formation of neutrophil extracellular traps.

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4-, and Ca++-dependent process†.

Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

Tissue-resident mast cells (MCs) are well known for their role in inflammatory responses and allergic and anaphylactic reactions, but they also contribute to processes of arterial remodeling. Although ribosomes and cytosolic RNAs are located around secretory granules in mature MCs, their functional role in MC responses remains unexplored. Previous studies by our group characterized extracellular RNA (eRNA) as an inflammatory and pathogenetic factor in vitro and in vivo. In the present study, RNA-containing MCs and eRNA were located in close proximity to growing collateral arteries in vivo. In vitro, various agonists were found to induce the degranulation of MCs and the concomitant release of eRNA in association with microvesicles (MVs). The liberation of eRNA from MCs was abolished by MC stabilizers or by preventing the increase of intracellular Ca2+ in MCs. eRNA was found to be mainly contained inside MVs, as demonstrated by electron microscopy and immunocytochemistry. The exposure to and the uptake of MC-released MVs by cultured endothelial cells increased their expression of cytokines, such as monocyte chemoattractant protein or IL-6, in a dose- and time-dependent manner. These results indicate that RNA-containing MC-derived MVs are likely to be involved in inflammatory responses, relevant, for example, to processes of vascular remodeling.-Elsemüller, A.-K., Tomalla, V., Gärtner, U., Troidl, K., Jeratsch, S., Graumann, J., Baal, N., Hackstein, H., Lasch, M., Deindl, E., Preissner, K. T., Fischer, S. Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosis.

Besnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.

Metabolic requirements of Besnoitia besnoiti tachyzoite-triggered NETosis.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.

In vitro examination of the thrombolytic efficacy of tenecteplase and therapeutic ultrasound compared to rt-PA.

BACKGROUND: Optimizing thrombolytic therapy is vital for improving stroke outcomes. We aimed to develop standardized thrombolysis conditions to evaluate the efficacy of tenecteplase (TNK) compared to the current gold standard rt-PA (alteplase), with and without additional ultrasound treatment. We also wanted to introduce a new analytical approach to quantify fibrin fiber density in transmission electron microscopy (TEM). METHODS: In vitro clots that are similar to ex vivo clots concerning their histological condition and their durability were generated from whole blood. For five treatment groups we compared relative clot weight loss (each n = 60) and fibrin fiber density in TEM (each n = 5). The control group (A) was treated only with plasma. Two groups were designated for each rt-PA (B + C) and TNK (D + E). Groups C and E were additionally treated with ultrasound. Dosages were 50 µg/ml for rt-PA and 30 µg/ml for TNK. Results were evaluated by using analyses of variance (ANOVA) and post-hoc t-tests. RESULTS: Weight loss was increased significantly for all groups compared to the control group. Both TNK groups showed significantly increased weight loss compared to their counterpart rt-PA group (p ≤ 0.001). For TEM only group D showed significantly decreased fibrin fiber density (p < 0.05) compared to both rt-PA groups. Ultrasound did not significantly increase dissolution of clots with either method (best p = 0.16). CONCLUSIONS: Tenecteplase dissolved clots more effectively than rt-PA with and without ultrasound. A higher sample size could provide more convincing results for TEM.

Antarctophthirus microchir infestation in synanthropic South American sea lion (Otaria flavescens) males diagnosed by a novel non-invasive method.

Antarctophthirus microchir is a sucking louse species belonging to the family Echinophthiriidae and has been reported to parasitize all species of the subfamily Otariinae, the sea lions. Former studies on this ectoparasite mainly required fixation, immobilization, or death of host species and especially examinations of adult male sea lions are still very rare. Between March and May 2018, adult individuals of a unique "urban" bachelor group of South American sea lions (Otaria flavescens) living directly in the city of Valdivia, Chile, were studied regarding their ectoparasite infestation status. For first time, a non-invasive method in the form of a lice comb screwed on a telescopic rod and grounded with adhesive tape was used for sample taking process. Overall, during combing different stages of A. microchir were detected in 4/5 O. flavescens individuals, especially at the junction between the back and hind flippers. Our findings represent the first report of A. microchir infesting individuals of this synanthropic colony and fulfilling complete life cycle in a sea lion group despite inhabiting freshwater and in absence of females/pups. Our "telescopic lice comb apparatus" offers a new strategy to collect different stages of ectoparasites and a range of epidermal material, such as fur coat hair and superficial skin tissue for a broad spectrum of research fields in wildlife sciences in an unmolested and stress reduced manner.

Müller glial cells of the primate foveola: An electron microscopical study.

Previous studies on the ultrastructure of the primate foveola suggested the presence of an inverted cone-like structure which is formed by 25-35 specialized Müller cells overlying the area of high photoreceptor density. We investigated the ultrastructure of the Müller cells in the foveola of a human and macaque retina. Sections through the posterior poles of an eye of a 40 years-old human donor and an eye of an adult cynomolgus monkey (Macaca fascicularis) were investigated with transmission electron microscopy. The foveola consisted of an inner layer (thickness, 5.5-12 µm) which mainly contained somata (including nuclei) and inner processes of Müller cells; this layer overlaid the central Henle fibers and outer nuclear layer. The inner layer contained numerous watery cysts and thin lamelliform and tubular Müller cell processes which spread along the inner limiting membrane (ILM). The cytoplasm of the outer Müller cell processes became increasingly dispersed and electron-lucent in the course towards the outer limiting membrane. The ILM of the foveola was formed by a very thin basal lamina (thickness, <40 nm) while the basal lamina of the parafovea was thick (0.9-1 µm). The data show that there are various conspicuous features of foveolar Müller cells. The numerous thin Müller cell processes below the ILM may smooth the inner surface of the foveola (to minimize image distortion resulting from varying light refraction angles at an uneven retinal surface), create additional barriers to the vitreous cavity (compensating the thinness of the ILM), and provide mechanical stability to the tissue. The decreasing density of the outer process cytoplasm may support the optical function of the foveola.

Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Polysialic Acid in Human Plasma Can Compensate the Cytotoxicity of Histones.

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

Clot Formation in the Presence of Acetylsalicylic Acid Leads to Increased Lysis Rates Regardless of the Chosen Thrombolysis Strategy.

BACKGROUND: Patients with acute ischemic strokes frequently take an acetylsalicylic acid (ASA) premedication. We determined the impact of ASA on different thrombolysis strategies in vitro. METHODS: For two clot types made from platelet-rich plasma (one with and one without ASA) lysis rates were measured by weight loss after 1 h for five different groups: in control group A clots were solely placed in plasma; in groups B and C clots were treated with rt-PA (60 kU/ml), and in groups D and E clots were treated with desmoteplase (DSPA; 2 µg/ml). Ultrasound (2 MHz, 0.179 W/cm2) was included in groups C and E. The fibrin mesh structures of the clots were investigated by electron microscopy. RESULTS: For both clot types lysis rates increased significantly for all treatment strategies compared to their control group (each p < 0.001). The addition of ASA significantly increased the lysis rate in all 5 groups (each p < 0.001) and led to a ceiling effect concerning the treatment. A semiquantitative analysis of transmission electron micrographs revealed a decreased fibrin density for clots with ASA. For both clot types DSPA and ultrasound led to a significant dissolution of the fibrin mesh (both p = 0.029). CONCLUSIONS: In vitro ASA pretreatment leads to significantly increased lysis rates due to a weaker fibrin mesh in platelet-rich plasma clots.

Myokardszintigraphie - eine Methode zur Erfassung der kardialen Komorbidität bei Psoriasis-Patienten?

HINTERGRUND: Kardiale Komorbiditäten bei Patienten mit Psoriasis stehen seit Jahren im Fokus. Ziel dieser Arbeit war es, im Rahmen einer Pilotstudie die Myokardszintigraphie als mögliche Früherkennungsmethode zu evaluieren. PATIENTEN UND METHODIK: Es wurden bei insgesamt 50 kardial asymptomatischen Patienten mit einer Psoriasis der Haut verschiedene Begleiterkrankungen erfasst. Dabei kam zur Erkennung von kardialem Risiko/ belastungsinduzierter Ischämie die Myokardszintigraphie zum Einsatz. ERGEBNISSE: Bei 28 Patienten (56 %) fanden sich pathologische Befunde in der Myokardszintigraphie. Davon zeigten 14 Patienten Zeichen einer sogenannten Small Vessel Disease (Kardiales Syndrom X). Darüber hinaus fanden sich weitere Begleiterkrankungen wie Adipositas, arterielle Hypertonie, Nikotinkonsum, Alkoholkonsum und erhöhte CRP-Werte. Die Häufigkeiten entsprachen im Wesentlichen den aktuellen Daten aus der Literatur. Wir konnten keinen signifikanten Zusammenhang von Schwere der Psoriasis oder der angegebenen Komorbiditäten mit einem pathologischen Befund in der Myokardszintigraphie feststellen. SCHLUSSFOLGERUNGEN: Mit der Myokardszintigraphie scheint ein sehr empfindliches, nicht invasives Früherkennungsverfahren zur Detektion kardialer Komorbidität bei Psoriasis-Patienten zur Verfügung zu stehen. Weitere größere Arbeiten mit Kontrollkollektiven und Kotrollmethoden, wie beispielsweise der Koronarangiographie, sind zur Überprüfung der Wertigkeit nötig.

Myocardial scintigraphy - a method for detecting cardiac comorbidity in psoriasis patients?

BACKGROUND: In recent years, cardiac comorbidities in psoriasis patients have increasingly moved into the focus of clinical research. The objective of the present study was to evaluate myocardial scintigraphy as a screening method in patients with psoriasis. PATIENTS AND METHODS: Assessment of various comorbidities in 50 psoriasis patients without clinical symptoms of cardiac disease. Myocardial scintigraphy was employed to detect cardiac risk/exercise-induced ischemia. RESULTS: Twenty-eight patients (56 %) had pathological findings on myocardial scintigraphy. Fourteen individuals showed evidence of small-vessel disease (cardiac syndrome X). Other comorbidities included obesity, arterial hypertension, nicotine and alcohol abuse, as well as elevated CRP levels. Frequencies largely corresponded to those reported in the recent literature. There was no significant correlation between the severity of psoriasis or any comorbidities and pathological findings on myocardial scintigraphy. CONCLUSIONS: Myocardial scintigraphy seems to be a very sensitive, noninvasive method for the early detection of cardiac comorbidities in psoriasis patients. However, determining its true diagnostic value will require larger studies with control subjects and control methods such as coronary angiography.

Thermal Impact and the Relevance of Body Size and Activity on the Oxygen Consumption of a Terrestrial Snail, Theba pisana (Helicidae) at High Ambient Temperatures.

Metabolism, mainly driven by oxygen consumption, plays a key role in life, as it is one of the main ways to respond to extreme temperatures through internal processes. Theba pisana, a widespread Mediterranean land snail, is exposed to a wide range of ambient temperature. In this species the oxygen consumption was tested as a response variable by multiple regression modelling on the "explanatory" variables shell-free mass, temperature, and relative humidity. Our results show that the oxygen consumption of T. pisana can be well described (73.1%) by these three parameters. In the temperature range from 23 °C to 35 °C the oxygen consumption decreased with increasing temperature. Relative humidity, in the range of 67% to 100%, had the opposite effect: if it increases, oxygen consumption will increase as well. Metabolism is proportional to an individual's mass to the power of the allometric scaling exponent α, which is between 0.62 and 0.77 in the mentioned temperature range. CT scans of shells and gravimetry revealed the shell-free mass to be calculated by multiplying the shell diameter to the third power by 0.2105. Data were compared to metabolic scaling exponents for other snails reported in the literature.

Human dendritic cell interactions with the zoonotic parasite Cryptosporidium parvum result in activation and maturation.

Cryptosporidiosis in humans is caused by infection of the zoonotic apicomplexan parasite Cryptosporidium parvum. In 2006, it was included by the World Health Organization (WHO) in the group of the most neglected poverty-related diseases. It is characterized by enteritis accompanied by profuse catarrhalic diarrhea with high morbidity and mortality, especially in children of developing countries under the age of 5 years and in HIV patients. The vulnerability of HIV patients indicates that a robust adaptive immune response is required to successfully fight this parasite. Little is known, however, about the adaptive immune response against C. parvum. To have an insight into the early events of the adaptive immune response, we generated primary human dendritic cells (DCs) from monocytes of healthy blood donors and exposed them to C. parvum oocysts and sporozoites in vitro. DCs are equipped with numerous receptors that detect microbial molecules and alarm signals. If stimulation is strong enough, an essential maturation process turns DCs into unique activators of naïve T cells, a prerequisite of any adaptive immune response. Parasite exposure highly induced the production of the pro-inflammatory cytokines/chemokines interleukin (IL)-6 and IL-8 in DCs. Moreover, antigen-presenting molecules (HLA-DR and CD1a), maturation markers, and costimulatory molecules required for T-cell stimulation (CD83, CD40, and CD86) and adhesion molecules (CD11b and CD58) were all upregulated. In addition, parasite-exposed human DCs showed enhanced cell adherence, increased mobility, and a boosted but time-limited phagocytosis of C. parvum oocysts and sporozoites, representing other prerequisites for antigen presentation. Unlike several other microbial stimuli, C. parvum exposure rather led to increased oxidative consumption rates (OCRs) than extracellular acidification rates (ECARs) in DCs, indicating that different metabolic pathways were used to provide energy for DC activation. Taken together, C. parvum-exposed human DCs showed all hallmarks of successful maturation, enabling them to mount an effective adaptive immune response.

Influence of different pre-treatments on the resin infiltration depth into enamel of teeth affected by molar-incisor hypomineralization (MIH).

OBJECTIVES: This in vitro pilot study aimed to evaluate whether different pre-treatments (demineralization, deproteinization, (chemo-)mechanical reduction of the surface layer) influence the penetration depth of a resin infiltrant into MIH-affected enamel compared to initial carious lesions. METHODS: Thirty extracted human permanent molars with non-cavitated initial carious lesions (n = 5) or MIH (n = 25) were chosen and randomly assigned to six experimental groups: IC: initial caries; M: MIH; MN: MIH, 5.25% sodium hypochlorite; MM: MIH, microabrasion; MA: MIH, air abrasion; MAN: MIH, air abrasion and 5.25% sodium hypochlorite. A modified indirect dual fluorescence staining method was adopted to assess the penetration depth (PD) of the resin infiltrant and the lesion depth (LD) by confocal laser scanning microscopy (CLSM). Exemplarily, scanning electron microscopic (SEM) images were captured. The relationship between group assignment and penetration/lesion depth was estimated using a linear mixed model incorporating the tooth as random effect (two observations/tooth). The significance level was set at p < 0.05. RESULTS: For MIH-affected molars, the mean PD (in µm; median, [minimum-maximum]) were M (178.2 [32.5-748.9]), MN (275.6 [105.3-1131.0]), MM (48.7 [0.0-334.4]), MA (287.7 [239.4-491.7]), and MAN (245.4 [76.1-313.5]). Despite the observed differences in PD between the groups, these could not be statistically verified (Bonferroni, p = 0.322). The percentage penetration was significantly higher for IC than for MIH groups (Bonferroni, p < 0.05). SIGNIFICANCE: Compared to IC, resin infiltration into MIH-affected enamel ist more variable. Different pre-treatments influence the resin penetration into developmentally hypomineralized enamel to a fluctuating level.

Tau oligomers impair artificial membrane integrity and cellular viability.

The microtubule-associated protein Tau is mainly expressed in neurons, where it binds and stabilizes microtubules. In Alzheimer disease and other tauopathies, Tau protein has a reduced affinity toward microtubules. As a consequence, Tau protein detaches from microtubules and eventually aggregates into ß-sheet-containing filaments. The fibrillization of monomeric Tau to filaments is a multistep process that involves the formation of various aggregates, including spherical and protofibrillar oligomers. Previous concepts, primarily developed for Aß and α-synuclein, propose these oligomeric intermediates as the primary cytotoxic species mediating their deleterious effects through membrane permeabilization. In the present study, we thus analyzed whether this concept can also be applied to Tau protein. To this end, viability and membrane integrity were assessed on SH-SY5Y neuroblastoma cells and artificial phospholipid vesicles, treated with Tau monomers, Tau aggregation intermediates, or Tau fibrils. Our findings suggest that oligomeric Tau aggregation intermediates are the most toxic compounds of Tau fibrillogenesis, which effectively decrease cell viability and increase phospholipid vesicle leakage. Our data integrate Tau protein into the class of amyloidogenic proteins and enforce the hypothesis of a common toxicity-mediating mechanism for amyloidogenic proteins.