In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.     

Mechanism of cadmium uptake by activated sludge

Summary The significance of metabolic activity in cadmium uptake by unacclimated activated sludge was studied. Below 30 mg/l cadmium in solution, biosorption was found to follow the Freundlich isotherm, which is the most common pattern for physico-chemical adsorption. More than 95% of total cadmium uptake was achieved within 5 min metal-sludge contact time. Biosorption increased strongly when the initial cadmium concentration in solution was raised from 10 to 100 mg/l, whereas in the same concentration range the metabolic activity of the sludge, as measured by respiratory activity and extracellular protein production, was very significantly inhibited. The addition of nutrients at low but significant levels failed to increase cadmium uptake in 2 h contact time, while in 24 h the addition of nutrients caused the biosorption to increase by only 5–10% without any significant growth of the biomass. Biosorption was found to increase with temperature between 5° C and 40° C, in correlation with an increase in the metabolic activity of the sludge. Pretreatment of the sludge with metabolic inhibitors (NaN₃ and UV rays) appeared to cause only a very slight decrease (5–10%) of biosorption. These results suggest that metabolic uptake of cadmium was low and that adsorption to the surface of the cells was the major mechanism of uptake.Offprint requests to: S. S. Sofer     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

Evidence for Na+/H+ antiport inMethanospirillum Hungatei

The growth and methane formation ofMethanospirillum hungatei were inhibited by an inhibitor of Na⁺/H⁺ antiport amiloride. After addition of NaCl or LiCl, when the cells had a lower intracellular pH and were deenergized, they extruded protons into the external medium. The acidification of the external medium was stimulated by protonophores and inhibited by amiloride. These findings suggest the existence of an Na⁺/H⁺ antiport in the cytoplasmic membrane ofM. hungatei and its role in the energetics of methanogenic bacteria.     

Divergent evolutionary processes associated with colonization of offshore islands

Oceanic islands have been a test ground for evolutionary theory, but here, we focus on the possibilities for evolutionary study created by offshore islands. These can be colonized through various means and by a wide range of species, including those with low dispersal capabilities. We use morphology, modern and ancient sequences of cytochrome b (cytb) and microsatellite genotypes to examine colonization history and evolutionary change associated with occupation of the Orkney archipelago by the common vole (Microtus arvalis), a species found in continental Europe but not in Britain. Among possible colonization scenarios, our results are most consistent with human introduction at least 5100 bp (confirmed by radiocarbon dating). We used approximate Bayesian computation of population history to infer the coast of Belgium as the possible source and estimated the evolutionary timescale using a Bayesian coalescent approach. We showed substantial morphological divergence of the island populations, including a size increase presumably driven by selection and reduced microsatellite variation likely reflecting founder events and genetic drift. More surprisingly, our results suggest that a recent and widespread cytb replacement event in the continental source area purged cytb variation there, whereas the ancestral diversity is largely retained in the colonized islands as a genetic ‘ark’. The replacement event in the continental M. arvalis was probably triggered by anthropogenic causes (land‐use change). Our studies illustrate that small offshore islands can act as field laboratories for studying various evolutionary processes over relatively short timescales, informing about the mainland source area as well as the island.     

Cognitive flexibility deficits in a mouse model for the absence of full‐length dystrophin

Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full‐length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full‐length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full‐length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.     

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.     

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p < 0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.